Laser-based fourier ptychographic imaging systems and methods

ABSTRACT

Certain embodiments pertain to laser-based Fourier ptychographic (LFP) imaging systems, angle direction devices used in the LFP imaging systems, optical switches used in the LFP imaging systems, and LFP imaging methods. The LFP systems include an angle direction device for directing laser light to a sample plane at a plurality of illumination angles at different sample times. The LFP systems also include an optical system and a light detector. The optical system receives light issuing from the sample being imaged and propagates and focuses the light to the light detector acquiring raw intensity images.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/165,084, titled “Laser Based Fourier Ptychographic Microscopy”and filed on May 21, 2015, which is hereby incorporated by reference inits entirety and for all purposes.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. OD007307and AI096226 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

Certain embodiments described herein are generally related to digitalimaging techniques, more specifically, in high-resolution imaging formicroscopy and photography in applications such as, for example,pathology, haematology, and semiconductor wafer inspection.

BACKGROUND

Imaging lenses ranging from microscope objectives to satellite-basedcameras are physically limited in the total number of features they canresolve. These limitations are a function of the point-spread functionsize of the imaging system and the inherent aberrations across its imageplane field of view. Referred to as the space-bandwidth product, thephysical limitation scales with the dimensions of the lens but isusually on the order of 10 megapixels regardless of the magnificationfactor or numerical aperture (NA). While conventional imaging systemsmay be able to resolve up to 10 megapixels, there is typically atradeoff between point-spread function and field of view. For example,certain conventional microscope objectives may offer a sharppoint-spread function across a narrow field of view, while othersimaging systems with wide-angle lenses can offer a wide field of view atthe expense of a blurry point-spread function.

Traditionally, the resolution of an image sensor, such as in a digitalcamera, determines the fidelity of visual features in the resultantimages captured by the image sensor. However, the resolution of anyimage sensor is fundamentally limited by geometric aberrations in thelens or lenses used to focus light onto the image sensor. This isbecause the number of resolvable points for a lens, referred to as theSBP, is fundamentally limited by geometrical aberrations. While CMOS andCCD technologies have been demonstrated having image sensors with pixelsin the 1 micron (μm) range, it remains a challenge to design andmanufacture lenses which have the resolving power to match theresolution of such image sensors.

Certain interferometric synthetic aperture techniques try to increasespatial-bandwidth product. Most of these interferometric syntheticaperture techniques include setups that record both intensity and phaseinformation using interferometric holography such as off-line holographyand phase-shifting holography. Interferometric holography has itslimitations. For example, interferometric holography recordingstypically use highly coherent light sources. As such, the constructedimages typically suffer from coherent noise sources such as specklenoise, fixed pattern noise (induced by diffraction from dust particlesand other optical imperfections in the beam path), and multipleinterferences between different optical interfaces. Thus the imagequality is typically worse than from a conventional microscope. On theother hand, using off-axis holography sacrifices spatial-bandwidthproduct (i.e., reduces total pixel number) of the image sensor. Inaddition, interferometric imaging techniques may be subject touncontrollable phase fluctuations between different measurements. Hence,accurate a priori knowledge of the sample location may be needed to seta reference point in the image recovery process. Another limitation isthat many of these interferometric imaging systems require mechanicalscanning to rotate the sample and thus precise optical alignments,mechanical control at a sub-micron level, and associated maintenancesare required by these systems. In terms of spatial-bandwidth product,these interferometric imaging systems may present little to no advantageas compared with a conventional microscope. Previous lensless microscopysuch as in-line holography and contact-imaging microscopy also presentdrawbacks. For example, conventional in-line holography does not workwell with contiguous samples and contact-imaging microscopy requires asample to be in close proximity to the sensor.

A high spatial-bandwidth product is very desirable in imagingapplications such as microscopy for biomedical imaging such as used inpathology, haematology, phytotomy, immunohistochemistry, andneuroanatomy. For example, there is a strong need in biomedicine andneuroscience to image large numbers of histology slides for evaluation.

SUMMARY

Certain embodiments pertain to laser-based Fourier ptychographic (LFP)imaging systems and their components. For example, some embodimentsrelate to LFP systems comprising an angle direction device, an opticalsystem comprising a collection element and a focusing element, and alight detector(s). The angle direction device is configured orconfigurable to direct laser light from a laser light source(s) to asample plane generally at a specimen surface. The laser light isdirected at a sequence of illumination angles at different sample times.The collection element is configured to receive light issuing from aspecimen when it is located on the specimen surface. The sequence ofillumination angles and numerical aperture of the collection elementcorrespond to overlapping regions in a Fourier domain. The lightdetector is configured or configurable to receive light focused by thefocusing element of the optical system and to acquire a plurality of rawintensity images of the specimen when it is located on the specimensurface and illuminated. Each raw intensity image acquired by the lightdetector(s) corresponds to a different illumination angle of thesequence of illumination angles. The LFP systems may also include aprocessor configured or configurable to execute instructions foriteratively updating overlapping regions in the Fourier domain with theplurality of intensity images acquired by the light detector to generatea high resolution image of the specimen. In some cases, the processor isalso configured or configurable to execute instructions for filteringout low spatial frequency artifacts associated laser light by using adifferential phase contrast deconvolution procedure.

Certain embodiments pertain to angle direction devices configured orconfigurable to direct laser light from a laser light source(s) to asample plane generally at a specimen surface of an LFP system.

In one embodiment, the angle direction device comprises a plurality offixed mirrors and one or more rotatable mirrors. Each fixed mirror isoriented to reflect laser light at one of the plurality of illuminationangles to the specimen surface. The one or more rotatable mirrors areconfigured or configurable to reflect laser light from the laser lightsource sequentially to different fixed mirrors of the plurality of fixedmirrors at different sampling times. The fixed mirrors are oriented toreflect laser light received from the one or more rotatable mirrors tothe specimen surface at the sequence of illumination angles.

In another embodiment, the angle direction device comprises a pluralityof optical fibers and one or more optical switches. Each of theplurality of optical fibers has a first and second end portion. The oneor more optical switches are in optical communication with a laser lightsource. The one or more optical switches are configured or configurableto switch at different sampling times to direct laser light from thelaser light source to the first end portion of different optical fibersof the plurality of optical fibers when the laser light source isactivated. The optical pathways are configured so that each second endportion directs laser light to one of the sequence of illuminationangles when the one or more optical switches is switched to thecorresponding optical fiber and the laser light source is activated.

In another embodiment, the angle direction device comprises a movablestage (e.g., X-Y stage) and an optical fiber coupled to the movablestage and optically coupled at one end to the laser light source. Themovable stage is configured or configurable to translate and/or rotatethe optical fiber to direct laser light from the other end of theoptical fiber to illuminate the specimen surface at the plurality ofillumination angles at the different sampling times.

In another embodiment, the angle direction device comprises one or morerotatable mirrors and a lens system. The one or more rotatable mirrorsare configured or configurable to direct the laser light from the laserlight source to the specimen surface through a lens system, the lenssystem configured such that rotation of the one or more rotatablemirrors causes the laser light to illuminate the specimen at thesequence of illumination angles.

In certain embodiments, the angle direction device comprises a surfaceand a plurality of fixed mirrors coupled to the surface, each fixedmirror oriented to receive laser light and reflect the laser light toone of the plurality of illumination angles.

Certain embodiments pertain to laser-based Fourier ptychographic imagingmethods employing at least one laser light source. The methods comprisedirecting laser light from a laser light source to a specimen surfacelocated at about a sample plane using an angle direction device at asequence of illumination angles. The methods further comprise receivinglight, at a light detector, issuing from a sample when the sample islocated on the specimen surface, the light received from an opticalsystem. The methods further comprise acquiring a plurality of intensityimages based on light received at the light detector, wherein eachintensity image corresponds to one of the illumination angles. Themethods further comprise constructing a higher resolution image bysimultaneously updating a pupil function and a sample spectrum, whereinthe sample spectrum is updated in overlapping regions with Fouriertransformed intensity images, wherein each of the overlapping regionscorresponds to one of the plurality of illumination angles.

These and other features are described in more detail below withreference to the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simplified block diagram of a laser-based Fourierptychographic (LFP) imaging system, in accordance with someimplementations.

FIG. 2 shows a schematic drawing of a cut-away view of an example of anLFP imaging system, in accordance with some implementations.

FIG. 3 shows a schematic drawing of a cut-away view of another exampleof an LFP imaging system, in accordance with some implementations.

FIG. 4 shows a schematic drawing of a cut-away view of another exampleof an LFP imaging system, in accordance with some implementations.

FIG. 5 shows a schematic drawing of a cut-away view of another exampleof an LFP imaging system, in accordance with some implementations.

FIG. 6 shows a schematic drawing of an orthogonal view of anotherexample of an LFP imaging system, in accordance with someimplementations.

FIG. 7A shows a schematic layout of an example of a mirror array, inaccordance with some implementations.

FIG. 7B shows a schematic layout of an example of a region in Fourierspace that corresponds to the mirror array of FIG. 7A, in accordancewith some implementations.

FIG. 8 shows a schematic drawing of an orthogonal view of anotherexample of an LFP imaging system, in accordance with someimplementations.

FIG. 9A shows a schematic drawing of an orthogonal view of anotherexample of a mirror array, in accordance with some implementations.

FIG. 9B shows a schematic drawing of an orthogonal view of anotherexample of a mirror array, in accordance with some implementations.

FIG. 10 shows a flowchart of an example of an LFP imaging method, inaccordance with some implementations.

FIG. 11 shows a flowchart depicting details of operations of an LFPimaging method that implements DPC deconvolution, in accordance withsome implementations.

FIG. 12 shows an example of a side-by-side comparison of LFPreconstructed images that have been enhanced with DPC deconvolution andLFP reconstructed images that have not been enhanced with DPCdeconvolution, in accordance with some implementations.

FIG. 13A shows an example of a raw image of a sample acquired by an LFPimaging system, in accordance with some implementations.

FIG. 13B shows an example of an LFP reconstructed high resolution imageof the sample of FIG. 13A, in accordance with some implementations.

FIG. 14 shows a schematic representation of a phase and amplitudereconstruction process during the performance of an LFP imaging method,according to an implementation.

DETAILED DESCRIPTION

Embodiments of the present invention will be described below withreference to the accompanying drawings. The following description isdirected to certain implementations for the purposes of describingvarious aspects of this disclosure. However, a person having ordinaryskill in the art will readily recognize that the teachings herein can beapplied in a multitude of different ways. Thus, the teachings are notintended to be limited to the implementations depicted solely in theFigures, but instead have wide applicability as will be readily apparentto one having ordinary skill in the art.

I. Introduction

Fourier ptychography imaging techniques generally involve synthesizingan image with a higher bandwidth using multiple low-bandwidth imagescaptured at different spatial frequency regions. Fourier ptychographytechniques provide resolution enhancement that can be implemented withan optical system to achieve resolution construction beyond the physicalcapabilities of the optical system alone. In certain cases, Fourierptychography imaging involves the acquisition of multiple raw intensityimages of a sample where each image is acquired using light at adifferent illumination angle than the others (variable illumination).

An imaging system that implements Fourier ptychography imagingtechniques using a microscope optical system is collectively referred toas an FPM. Some examples of existing FPMs that use light-emitting diode(LED) illumination can be found in G. Zheng, R. Horstmeyer and C. Yang,“Wide-field, high-resolution Fourier ptychographic microscopy,” NaturePhotonics, 2013, X. Ou, R. Horstmeyer, C. Yang and G. Zheng,“Quantitative phase imaging via Fourier ptychographic microscopy,”Optics Letters, 2013, X. Ou, R. Horstmeyer, G. Zheng and C. Yang, “Highnumerical aperture Fourier ptychography: principle, implementation andcharacterization,” Optics Express, 2015, “A phase space model of Fourierptychographic microscopy,” Opt. Express 22(1), 338-358 (2014), X. Ou, G.Zheng, and C. Yang, “Embedded pupil function recovery for Fourierptychographic microscopy,” Opt. Express 22(5), 4960-4972 (2014), X. Ou,R. Horstmeyer, G. Zheng, and C. Yang, “Counting White Blood Cells from aBlood Smear Using Fourier Ptychographic Microscopy,” PLoS One 10(7),e0133489 (2015), A. Williams, J. Chung, X. Ou, G. Zheng, S. Rawal, Z.Ao, R. Datar, C. Yang, and R. Cote, “Fourier ptychographic microscopyfor filtration-based circulating tumor cell enumeration and analysis,”J. Biomed. Opt. 19(6), 066007 (2014), and R. Horstmeyer, X. Ou, G.Zheng, P. Willems, and C. Yang, “Digital pathology with Fourierptychography,” Comput. Med. Imaging Graphics 42, 38-43 (2015), all ofwhich are hereby incorporated by reference for the discussion regardingFPMs.

As introduced above, existing implementations of FPMs typically used anarray of average consumable light-emitting diodes (LEDs) to provideangularly varying illumination. The time need to acquire the rawintensity images and generate a high resolution image with the existingLED-based FPMs was typically relatively slow since LEDs provide lowintensity illumination that required lengthy exposure times. Althoughhigh power LEDs could be used, they require cooling systems that made itdifficult to design a compact system.

Various aspects described here relate generally to Laser-based Fourierptychographic (LFP) imaging systems, devices, and methods that use aguided laser beam as illumination. In these aspects, the LFP imagingsystem includes an angle direction device to direct laser lightillumination at different illumination angles. For example, certain LFPimaging systems discussed herein are configured to enable highresolution imaging of a sample (also referred to herein as a “specimen”)on a specimen surface at a sample plane, using an angle direction deviceconfigured or configurable to direct laser light sequentially atdifferent illumination angles to the sample plane. The use of a laserlight sometimes present speckles during image acquisition due toreflections between glass surfaces in the system. If not mitigated,these speckles would appear as slowly varying background fluctuations inthe final reconstructed image. The LFP imaging method is adapted tomitigate artifacts from any speckles.

Since LFP imaging systems use laser light, they have several advantagesas compared to existing Fourier ptychography imaging systems that useaverage consumable LED array to provide variable illumination. First,laser light has brighter illumination as compared to conventional LEDswhich enables shortening the exposure time of raw intensity imageacquisition. For example, certain LFP systems have exposure times in therange of about 10 microseconds to about 100 milliseconds per frame ascompared to LED-based systems which have exposure times in the range of10 millisecond to 10 seconds per frame that typically use averageconsumable LEDs with relatively low power. Shorter exposure time canprovide an imaging scheme with higher temporal resolution suitable forresolving transient biological processes. Since the duration of animaging cycle is mostly driven by the time intensive raw imageacquisition process, using laser light significantly reduces the cycletime (e.g., range of 100 milliseconds to less than about 10 seconds) ascompared to LED-based systems (e.g., range of more than 10 seconds to1000 seconds). Additionally the broader range and finer spectrum of alaser light source potentially allows for spectral imaging capability.Conventional LED-based systems used LEDs with a broad spectrum and alimited number of choices for the central wavelength. Thus, thepotential application of conventional LED-based Fourier ptychographysystems was limited to the spectra offered by the LEDs.

As introduced above, various aspects described herein relate to LFPimaging systems that implement LFP methods to obtain high-resolutionimages of a sample. In certain implementations, LFP imaging systemsinclude a specimen surface for receiving a sample being imaged, an angledirection device, an optical system, and a light detector such as animage sensor. The angle direction device is configured or configurableto direct laser light from a laser light source to the specimen surfaceat a sequence of illumination angles. The optical system comprises acollection element (e.g., lens) for receiving light issuing from thesample when it is illuminated from laser light directed by the angledirection device. The optical system also includes a focusing element(e.g., lens) for focusing light propagated from the collection elementto the light detector. In some cases, the LFP imaging system furtherincludes a controller in communication with the angle direction deviceand/or laser light source and configured to cause laser light from thelaser light source to be directed by the angle direction device to thespecimen surface at the sequence of illumination angles at particularsampling times. The controller may also be in communication with thelight detector to synchronize exposure time of the light detector formeasuring the intensity distribution data of each raw intensity imagewhile laser light is illuminating from one of the sequence ofillumination angles. Intensity distribution data is measured at thelight detector when the sample is illuminated at each of theillumination angles. The intensity distribution data measured at thelight detector when the sample is illuminated at a particularillumination angle is referred to herein as an “intensity image” or “rawimage” corresponding to the particular illumination angle. A sequence ofintensity images of the sample is acquired over the course of an entire“image acquisition phase” during which the sample is sequentiallyilluminated at different illumination angles. The optical system focuseslight scattered by the sample onto a light detector. Over the course ofthe image acquisition phase, the light detector takes a sequence ofintensity distribution measurements (intensity images), one rawintensity image being measured for each illumination angle. A processorcombines the raw intensity images in the spatial frequency domain usinga reconstruction process to correct aberrations and to render a singlehigh-resolution image of the sample.

Since the laser light is generally brighter than light from standardLEDs, holding the sensitivity of the light detector constant, use of alaser light source allows for a faster image acquisition phase than ifstandard LEDs were used. The LFP imaging systems and methods describedherein allow for a compact imaging system that is able to acquire asufficient number of intensity images to generate a high resolutionimage of a sample over a short image acquisition phase, e.g. potentiallyless than 1 second. In one embodiment, an LFP system can be configuredto generate a high resolution image of a sample in less than 1 second.In another embodiment, an LFP system can be configured to generate ahigh resolution image of a sample in less than 10 seconds. In anotherembodiment, an LFP system can be configured to generate a highresolution image of a sample in less than 60 seconds. In one embodiment,an LFP system can be configured to generate a high resolution image of asample in a range of 1 to 100 seconds. In one embodiment, an LFP systemcan be configured to acquire an intensity image at a rate of 100 persecond. In one embodiment, an LFP system can be configured to acquire anintensity image at a rate of 1000 per second.

The LFP imaging systems disclosed herein have additional benefitscompared to e Fourier ptychography imaging systems that use standardLEDs. By way of example, as discussed above, certain LFP imaging systemsdescribed herein are capable of capturing sufficient intensity images togenerate a high resolution image of a specimen in a relatively shortamount of time. Therefore, unlike slower FP imaging systems that usestandard LEDs, LFP imaging systems may more effectively image movingspecimens.

Some examples of LFP imaging systems are described in further detailbelow with reference to the Figures.

I. Laser-Based Fourier Ptychographic (LFP) Systems

A. LFP Systems

FIG. 1 is a block diagram of an LFP imaging system 100 capable ofimplementing an LFP imaging method, according to embodiments. At a highlevel, the LFP imaging system 100 is configured or configurable tosequentially illuminate a sample being imaged by laser light directed atdifferent illumination angles, to capture a sequence of intensity imageswhere each intensity image is captured when the sample is illuminated bylaser light directed at one of the illumination angles, and process theraw image data to reconstruct a full resolution complex image of thesample while correcting for any aberrations from, for example, specklesgenerated through the use of laser light.

The LFP imaging system 100 includes a laser illumination system 110, anoptical system 120, an imaging system 130, a controller 140, acommunication interface 150, and a display 152 in communication with thecommunication interface 150, and internal memory 160. The LFP imagingsystem 100 also optionally (denoted by dashed line) includes acommunication interface 170 and an external computing device or system172 in communication with the communication interface 170. The LFPimaging system 100 also optionally includes a communication interface180 and an external memory device or system 182 in communication withthe communication interface 180 for optional storage of data to theexternal memory device or system 182. The LFP imaging system 100 alsooptionally includes a network communication interface 190 and anexternal network 192 (for example, a wired or wireless network)communication with the communication interface 190. Each of thecommunication interfaces 150, 160, 170, 180, 190 is in communicationwith the controller 140. The laser illumination system 110 is configuredor configurable to provide laser light illumination from a sequence ofillumination angles. The optical system 120 generally has one or morelenses that propagate light issuing from the sample to one or more lightdetectors of the imaging system 130. The controller 140 is incommunication with the internal memory 160 to retrieve and store data tothe internal memory 160. The controller 140 is configured orconfigurable to output raw image data, processed image data, and/orother data over a communication interface 150 for display on a display152. The controller 140 is in communication with the imaging system 130which is in communication with the optical system 120. The describedcommunication between components of the LFP imaging system 100 may be inwired form and/or wireless form.

The controller 140 comprises one or more processors which are incommunication with internal memory 160 and/or other computer readablememory (CRM). The controller 140 controls operations of the LFP imagingsystem 100 based on instructions stored in memory (e.g., internal memory160). The controller 140 is in electrical communication with the imagingsystem 130 to receive the raw image data from the imaging system 130.Optionally (denoted by dotted lines), the controller 140 may also be inelectrical communication with the laser illumination system 110 tocontrol illumination, for example, in order to synchronize theillumination with the exposure times during acquisition of intensityimages by the imaging system 130. The controller 140 or anotherprocessor controls the illumination from laser light source(s). Forexample, the controller 140 or another processor may control theoperation of an angle direction device that directs light from the laserlight source(s) to at predefined illumination angles at particular timesand for particular durations during various image acquisition exposuresand/or control the activation of laser light source(s), for example, bypowering on during the image acquisition phase. In some implementations,the controller 140 is further configured to execute instructions toperform processing operations on the raw image data such as operationsperformed as part of the LFP imaging method.

The imaging system 130 is in communication with the optical system 120to receive light from the optical system 120 and capture raw imageswhile the sample is on the specimen surface and illuminated, each rawimage captured over an exposure time. The laser illumination system 110is in communication with the optical system 120 to provide laser lightto a sample being imaged on the specimen surface such that lightscattered by or otherwise issuing from the sample is propagated throughthe optical system 120 to light detector(s) of the imaging system 130which capture the raw images. When laser light illuminates the sample,light scattered or otherwise issuing from the sample is propagatedthrough the optical system 120 to the imaging system 130 which capturesa sequence of intensity images.

The LFP imaging systems described herein include an angle directiondevice. An angle direction device generally refers to one or morecomponents that are configured or configurable to guide a laser beam tothe sample plane at N different illumination angles at N different imagesampling times. During the image acquisition phase, the sample beingimaged is located on the specimen surface at the sample plane and thelaser light is directed to the sample being imaged. In certainembodiments, N has a value in a range of between 2 to 1000. In one case,N has a value in a range of between 90 and 200. In one case, N=95. Inone case, N is less than 100. In one case, N is less than 200. In onecase, N has a value in a range of between 50 and 100. In one case, N hasa value in a range of between 2 and 100. In one case, N has a value in arange of between 100 and 200.

Generally the angle direction device is designed to guide the laser beamto the sample plane to provide oblique illumination. The angle directiondevice is typically optically coupled with (or includes) one or morelaser light sources. The angle direction device also includes one ormore devices for directing the laser beam from the laser light source tothe specimen surface at the N different illumination angles described inthe preceding paragraph. For example, the angle direction device mayinclude one or more mirrors and/or movement mechanisms to direct thelaser beam sequentially to the different illumination angles at theappropriate sampling times. In one embodiment, the angle directiondevice includes an optical port for receiving an optical fiber coupledwith the laser light source(s) and is able to translate and/or rotatethe optical fiber to provide the oblique illumination described above.In one implementation, the angle direction device includes multipleoptical ports for receiving multiple optical fibers. In this case, theangle direction device may also include one or more optical switches forswitching laser light from the laser light source to a single opticalfiber. In another implementation, the angle direction device may includeone or more rotatable mirrors that direct light from the laser lightsource by way of an array of fixed mirrors, each of which may bepositioned to illuminate the specimen surface at one of the illuminationangles. In yet another implementation, the angle direction device mayinclude one or more rotatable mirrors that direct light from the laserlight source to the specimen surface by way of a system of lenses thatrefract the light from the laser light source to illuminate the specimensurface at each of the illumination angles.

The angle direction device is generally configured or configurable tocause light from the laser light source to illuminate the sample planeat the specimen surface at different angles sequentially. The image datafrom the sequence of images captured based on illumination at differentillumination angles corresponds to overlapping regions in Fourier space.The angle direction device is typically configured to direct laser lightto illuminate at a sequence of illumination angles that provide for anoverlapping area of neighboring regions of image data in Fourier spacewhere the overlapping area is of at least a certain minimum amount (e.g.65% overlap, 75% overlap, 70% overlap, 80% overlap, in a range of10%-65% overlap, in a range of 65%-75% overlap, etc.). To provide thisminimum amount of overlap of neighboring regions in Fourier space, theangle direction device may have elements that are configured so that thedifference between adjacent illumination angles is less than a certainmaximum angular difference.

The laser light source(s) may be a component of or may be separate fromthe LFP system 100. The intensity of light provided by the laser lightsource may vary, for example, depending on the type of sample beingimaged. By way of example, dead skin samples can tolerate a power perunit area of up to several W/cm². On the other hand, non-biologicalsamples may be able to tolerate a much higher power per unit areawithout being degraded. Although examples described herein typicallydiscuss a laser light source that provides visible light e.g. some ofthe LFP imaging systems disclosed herein generally operate with a laserlight source with a wavelength in the visible range between 400 nm and750 nm, the disclosure is not so limiting. In other embodiments, thelaser wavelength may be in the ultra-violet region or in the infra-redregion and the focusing element and glass surfaces of the LFP systemwould need to be appropriately substituted to work in these differentwavelength ranges. In addition, other sources of laser radiation may beused, e.g. a maser, a free electron laser, an x-ray laser, an acousticlaser, etc. When different types of lasers are used, many of the opticalelements, e.g. lenses or mirrors, disclosed herein may be substitutedwith suitable alternatives. By way of example, where an X-ray ormicrowave laser is used, mirrors and/or lenses that are configured toreflect or refract x-rays or microwaves respectively may be included assubstitutes for lenses and or mirrors described and/or depicted infigures herein.

The LFP systems described herein include an optical system that isgenerally configured to propagate illumination issuing from the sampleto the one or more light detectors to be able to capture raw images ofthe sample. The optical system comprises at least a collection elementand a focusing element. The collection element (e.g., lens) of an LFPsystem comprises one or more lenses and is designed to collect lightscattered by or otherwise issuing from the specimen at the sample plane.Some examples of suitable collection elements include an f=50 mm lens(e.g., f/1.8 D AF Nikkor), 4× Olympus Plan Achromat objective 0.10 NA,CFI Plan Achromat 10× objective NA 0.25, an Valumax objective lens 5×0.10 NA, and the like. The focusing element comprises one or more lensesand is located to receive incident light from the optical system andfocus the light to the light detector. An example of suitable focusingelements include a tube lens (e.g., a f=200 mm tube lens such as theThorlabs® ITL200 tube lens). Although certain illustrated examples areshown with the collection and focusing elements arranged in a 4farrangement, it would be understood that the disclosure is not solimiting and that other arrangements can be used. For example, thecollection and focusing elements can be arranged in a 6f arrangement. Inother examples, other optical elements such as bandpass filters, beamsplitters, and/or mirrors can be included.

In some embodiments, the optical system of an LFP system has an irislocated at the back focal plane of the collection element of the opticalsystem. An iris generally refers to a circular aperture that can beadjusted in diameter to limit the amount of light collected by theoptical system. Reducing the diameter and blocking out the outer regionof light is equivalent to reducing the NA of the collection element,which means blocking out high spatial frequency information of thesample. Placing the iris is useful to accurately define the NA of theimaging system for the purpose of the LFP imaging method to preventaliasing in the detected images, etc. There are different ways tocontrol the size and shape of the iris. In one example, one can use amechanical iris which has its diameter adjustable with a slider (e.g.Ring-actuated SM2 iris diaphragm from Thorlabs). As another example, aniris may be a transmissive liquid crystal display (LCD) that can adjustthe contrast of its pixel elements to produce an aperture. As anotherexample, light may be guided to an SLM or DMD and then only a part ofthe beam reflected back into the system's optical path by turning ondifferent elements on the SLM or DMD. With the use of an LCD, SLM, andDMD, the iris's shape is not limited to a circle because any discretizedshape can be displayed on these displays and thus, the shape and sizemay be defined as the operator desires.

In some cases, the collection element, focusing element, illuminationelements, and other components of the LFP system may be selected basedon the particular imaging implementations. For example, when imaging ahistological sample, the appropriate system NA (objectiveNA+illumination NA) provided by selecting a particular collectionelement and illumination elements may be in the range of 0.5-0.75. Theobjective NA may be 0.05-0.2 and the illumination NA 0.4-0.7 to providethis system NA, for example. On the other hand, if a blood smear isbeing analyzed for malaria, a system NA in the range of 1.2-1.5 may bedesired.

Generally, the LFP systems described herein includes one or more lightdetectors. In certain examples, such as the one shown in FIG. 1, the LFPsystem includes an imaging system 130 comprising one or more lightdetectors. According to certain implementations, a light detector isconfigured to capture light and output a data signal including imagedata representative of the measured intensities of light received atparticular locations of the active surface of the light detector(referred to herein as a “light intensity distribution,” “intensitydistribution,” or simply as an “intensity image,” “raw intensity image,”“image,” or “image frame”). The image data output by the lightdetector(s) is transmitted (or “sent” or “communicated”) in a signal toa processor of, for example, a controller such as controller 140. Thelight detector(s) of an LFP imaging system can acquire a sequence of Nraw intensity images during the image acquisition process. Each lightdetector acquires an intensity image by measuring an intensitydistribution of light incident on the sensing area of light detectorduring an exposure time. Some examples of suitable light detectors areCMOS sensors, a charge-coupled device (CCD), and other similar imagingdevices. In one example, a light detector is a CCD having a pixel sizeof 5.5 μm such as the Prosilica GX6600 light detector. In certainimplementations, the one or more light detectors in an LFP system aremonochromatic light detectors.

The exposure time refers to the duration of time during which the lightdetector(s) measures a light intensity distribution of light received atits active surface to capture a single raw intensity image. The exposuretime used to capture an intensity image by the light detector(s) ofvarious implementations of the LFP systems depends on both thesensitivity of the light detector(s) and the intensity of the laserlight from the one or more laser source(s). In other words, increasingthe sensitivity of the light detector(s) and/or the intensity of thelaser light source decreases the exposure time. As such, the LFP imagingsystems described herein may be configured for particular exposure timesbased on the laser light source used. For example, a range of exposuretimes from 100 μs to 10 ms may be used for an LFP imaging system with alight detector in the form of a sCMOS detector and with a laser lightsource having a range of light intensities from 100 μW to 10 mW.

Each of the LFP systems described herein is configured to implement anLFP imaging method. Each imaging cycle of the LFP imaging methodgenerally includes a raw intensity image acquisition phase, ahigh-resolution reconstruction phase, and an optional display phase.During the raw image acquisition phase, light generated by the laserlight source(s) and directed by the angular direction device illuminatesthe sample plane at different illumination angles sequentially. Thesample is typically located on the specimen surface approximatelylocated at the sample plane during the image acquisition phase. Laserlight incident on the sample is scattered by the physical features ofthe sample as it passes through the sample. A portion of the scatteredlight then passes to the collection element of the optical system. Thefocusing element of the optical system focuses the scattered light fromthe sample to one or more light detectors. The one or more lightdetectors capture a sequence of raw intensity images during sequentialillumination of the sample with laser light at the sequence ofillumination angles. The processor(s) (e.g., processor of a controller)sends control signals to various system components to control theiroperations. The processor(s) controls the operations of the angulardirection device and/or the light detectors by sending control signals.

During the high-resolution reconstruction phase, the processor(s)processes the raw image data from the image acquisition phase togenerate processed image data. In some implementations, the processor(s)are configured or configurable to perform LFP processing operations onthe image data of a sequence of intensity images. In these cases, theprocessor(s) interpret raw image data from the sequence of acquiredintensity images, transform the relatively low-resolution raw intensityimage data into Fourier space, iteratively update the transformed rawimage data in Fourier space to reconstruct amplitude and phase data fora single high-resolution image of the sample and the pupil function ofthe LFP imaging system. In some cases, the one or more processors use adifferential phase contrast (DPC) deconvolution procedure to acquire thequantitative phase of the sample and use the phase data to correct fornoise in the captured images such as those caused by speckles that maybe caused by laser light illumination. During the optional displayphase, display data is sent to a display such as display 152 to displaydata such as the high-resolution image and other data.

In certain implementations, the processor(s) of an LFP system may beconfigured to perform parallel image processing such as, for example,when processing multiple tile images (i.e. portions of the full field ofview image) to reconstruct phase and amplitude data for multiple tileimages in parallel. These tile images can then be combined to form thefull field of view image. To perform parallel image processing, thecontroller would be configured to include at least one processor (or“processing unit”). Some examples of processors that can be used toperform parallel processing or non-parallel processing include, forexample, a general purpose processor (CPU), an application-specificintegrated circuit, an programmable logic device (PLD) such as afield-programmable gate array (FPGA), or a System-on-Chip (SoC) thatincludes one or more of a CPU, application-specific integrated circuit,PLD as well as a memory and various interfaces.

Returning to FIG. 1, the controller 140 is in communication withinternal memory device 160. The internal memory device 160 can include anon-volatile memory array for storing processor-executable code (or“instructions”) that is retrieved by the processor to perform variousfunctions or operations described herein for carrying out variousoperations on the image data. The internal memory device 160 also canstore raw and/or processed image data. In some implementations, theinternal memory device 160 or a separate memory device can additionallyor alternatively include a volatile memory array for temporarily storingcode to be executed as well as image data to be processed, stored, ordisplayed. In some implementations, the controller 140 itself caninclude volatile and in some instances also non-volatile memory.

In FIG. 1, the controller 140 is in communication with the communicationinterface 150 which is in communication with the display 152. In certainimplementations, the controller 140 is configured or configurable by anoperator to output raw image data or processed image data over thecommunication interface 150 for display on the display 152. In someimplementations, the controller 140 also can be configured orconfigurable by to output raw image data as well as processed image data(for example, after image processing) over a communication interface 170to an external computing device or system 172. Indeed, in someimplementations, one or more of the LFP operations can be performed bysuch an external computing device 172. In some implementations, thecontroller 140 also can be configured or configurable by a user tooutput raw image data as well as processed image data over acommunication interface 180 for storage in an external memory device orsystem 182. In some implementations, the controller 140 also can beconfigured or configurable by a user to output raw image data as well asprocessed image data (for example, after image processing) over anetwork communication interface 190 for communication over an externalnetwork 192 (for example, a wired or wireless network). The networkcommunication interface 190 also can be used to receive information suchas software or firmware updates or other data for download by thecontroller 140. In some implementations, the LFP imaging system 100further includes one or more additional interfaces such as, for example,various Universal Serial Bus (USB) interfaces or other communicationinterfaces. Such additional interfaces can be used, for example, toconnect various peripherals and input/output (I/O) devices such as awired keyboard or mouse or to connect a dongle for use in wirelesslyconnecting various wireless-enabled peripherals. Such additionalinterfaces also can include serial interfaces such as, for example, aninterface to connect to a ribbon cable. It should also be appreciatedthat the laser illumination system 110 can be electrically coupled tocommunicate with the controller 140 over one or more of a variety ofsuitable interfaces and cables such as, for example, USB interfaces andcables, ribbon cables, Ethernet cables, among other suitable interfacesand cables.

In some embodiments, the data signals output by the light detectors ofan LFP system may be multiplexed, serialized or otherwise combined by amultiplexer, serializer or other electrical component of the imagingsystem before being communicated to the controller. In certainimplementations, the controller can further include a demultiplexer,deserializer or other device or component for separating the image datafrom each of the light detectors so that the image frames can beprocessed in parallel by the controller or for separating the image datafrom each tile image so that the sequence of image frames of each tileimage can be processed in parallel by the controller.

Some examples of LFP systems are shown in FIGS. 2-6. Some of these LFPsystems have certain components that are similar to those of the LFPsystem 100 in FIG. 1 above.

B. LFP Systems with a Scanning Fiber Port

Certain implementations of an LFP imaging system include an angledirection device with a scanning fiber port. In these implementations,laser light is coupled to one end of an optical fiber and the other endof the optical fiber is attached with a fiber connector or directly toan optical port (also called output port) in a movable stage. Themovable stage is configured to translate the end of the optical fiber ina plane and/or rotate the end of the optical fiber. An example of amovable stage is a two-axial motorized stage such as an X-Y stage. Themovable stage is generally configured to move (also referred to as scan)the optical fiber to provide the sample plane with angularly varyingillumination from a sequence of illumination angles.

FIG. 2 is schematic drawing of an example of an LFP imaging system 200with a scanning fiber port, in accordance with some implementations. Anoptical fiber 201 is shown with an end 202 within a fiber connector 203coupled to an optical port 205 in a movable stage 212. Although shownwith a fiber connector 203, it would be understood that the opticalfiber 201 could be placed directed in the optical port 205 in anotherembodiment. The other end (not shown) of the optical fiber 201 isoptically coupled with a laser light source(s) and receives laser lightfrom the laser light source(s) when activated. In the illustratedexample, the laser light source(s) is activated and laser light 204spreads conically from the end 202 proximal to the fiber connector 203.A transparent layer 207 with a specimen surface 208 is also shown. Thespecimen surface 208 can receive a sample (not shown) being imaged. Insome cases, one or both of the fiber connector 203 and the optical fiber201 are components of the LFP imaging system 200. In other cases, thefiber connector 203 and optical fiber 201 are separate components fromthe LFP imaging system 200.

The LFP imaging system 200 comprises an angular direction device 212having a movable stage 214 with an optical port 205 configured toreceive the optical fiber 201. With the optical fiber 201 coupled to theoptical port 205, the movable stage 214 is configured or configurable tomove (translate/rotate) the optical fiber 201 to direct illumination ata plurality of illumination angles to the sample plane. The LFP imagingsystem 200 further comprises an optical system 220 having a collectionelement 222 and a focusing element 224, and a light detector 232 forreceiving light propagated by the optical system 220. Although theillustrated example shows the movable stage 214 as having an opticalport 205 in the form of a circular aperture, it would be understood thatother receiving configurations for the optical port 205 could be used. Aseries of arrows are depicted in FIG. 2 to show an example of thedirection of translational movement of the optical fiber 201 during animage acquisition phase of one implementation. Other implementations mayinclude other translation patterns or may include rotational movement aswell. In yet other implementations, the movable stage 214 may beconfigured for two axis rotational movement.

In one embodiment, the LFP imaging system 200 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

The LFP Imaging system 200 also includes an optical system 220 having acollection element 222 (e.g., lens) having a focal length, f₁, and afocusing element 224 (e.g., lens) having a focal length, f₂. Thecollection element has an objective NA and is configured to receivelight issuing from a specimen when it is located on the specimen surface207. The focusing element 224 is configured to focus light propagatedfrom the collection element 222 to the light detector 232. The sequenceof illumination angles and the objective NA correspond to overlappingregions in the Fourier domain, as described in further detail below. Inthe illustration, the optical system 220 is in a 4f arrangement wherethe collection element 222 is located so that the sample plane is f₁apart from it. The sample's Fourier plane is located f₁ away on theother side of 222. Fourier plane of the sample at the sample plane atspecimen surface 208 is a focal length, f₂, away from the focusingelement 224 and the focusing element 224 is located a distance of afocal length, f₂, away from the light detector. Other arrangements canbe used. For example, a 6f arrangement can be used. As another example,other optical elements (e.g., mirrors, bandpass filters, beam splitters,etc.) can be introduced into the optical path for other arrangements.

The LFP imaging system 200 further comprises a light detector 232 witheight discrete light elements. Although shown with eight discrete lightelements, it would be understood that other numbers of light elementscould be used. The collection element 222 is configured to receive lightissuing from the sample (not shown) being illuminated on the specimensurface 208 and the focusing element 224 focuses light propagated fromthe collection element 222 to the light detector 232.

While laser light 204 issuing from the optical fiber 201 is depicted asspreading conically outwards with a particular width, the width of thiscone depends on the NA of the optical fiber 201. Different types ofoptical fibers may be used to achieve the desired illumination. By wayof example, a single mode fiber optic with an NA in the range of 0.1-0.4may be used to achieve a narrower cone. In another example, a multi-modefiber with an NA in the range of 0.1 to 0.8 may be used to achieve awider cone.

The movable stage 214 may be any kind of stage to which the opticalfiber 201 with or without the fiber connector 203 can be attached andthat can be configured to translate and/or rotate the optical fiber 201to direct the optical fiber 201 to provide a laser beam to a sampleplane at a specimen surface 208 at a sequence of illumination angles asdescribed in the section above in the context of the FIG. 1. Forinstance, at a particular sampling time, the movable stage 214 may causethe optical fiber 201 to translate/rotate to a particularposition/direction at which laser light from the optical fiber 201illuminates a sample plane at the specimen surface 207 at a particularone of the illumination angles in the sequence of illumination angles.Once the fiber connector 203 has illuminated the sample plane at theparticular illumination angle for at least the exposure time such that alight detector 232 may acquire an intensity image, the movable stage 214may cause the optical fiber 201 to translate/rotate to anotherposition/direction at which the optical fiber 201 provides the laserbeam to the sample plane at another one of the illumination angles. Theoptical fiber 201 may remain at each position/direction for at least theexposure time of the light detector 232 for generating an intensityimage. This raw image acquisition phase may continue until the samplehas been illuminated at all of the illumination angles at the differentsampling times.

The movable stage 214 may cause optical fiber 201 to translate and/orrotate in the positions described in the preceding paragraph in avariety of manners. In the illustrated example, the movable stage 214 isconfigured to cause the optical fiber 201 to translate linearly in thex-direction and then shift to different locations in the y-direction inorder to scan back and forth in the X-Y plane. In this manner, theoptical fiber 201 provides a laser beam to illuminate the sample from Ndifferent positions in the X-Y plane along the arrows in FIG. 2.Alternatively, the movable stage 214 may be configured to cause theoptical fiber 201 to translate spirally outwards such that the opticalfiber 201 may illuminate the sample from N positions along a spiralmoving to successive points in an r-θ plane. In one implementation, themovable stage 214 is an X-Y stage.

The movable stage 214 may be controlled in a variety of manners. By wayof example, a processor (e.g. a processor of the controller 140 ofFIG. 1) or processors may be in communication with movable stage 214.The processor may retrieve instructions stored in a memory (e.g.internal memory 160 of FIG. 1) and the instructions may be executable tocause the movable stage 214 to translate and/or rotate such that theoptical fiber 201 is positioned/directed to provide the laser light tothe sample sequentially at different illumination angles at differentsampling times. In some case, the movable stage 214 holds the opticalfiber 201 at the position for at least the duration of the exposure timeused acquire an intensity image by the light detector 232.Alternatively, the movable stage 214 may have an internal memory andprocessor and may be programmed to cause the fiber connector 203 tofollow a pre-determined path.

It would be understood to those skilled in the art that the fiberconnector 203 may have shapes other than the one illustrated in FIG. 2.In addition, it would be understood that the optical fiber 201 may bedirectly attached to the movable stage 214 or may be attached throughthe fiber connector 203 to the movable stage 214.

At the instant in time illustrated in FIG. 2, the angle direction device212 is illuminating a sample (not shown) on the specimen surface 208.Generally, light incident on the specimen is scattered by the physicalfeatures of the specimen as it passes through the specimen. Thecollection element 222, which have a particular objective NA, receiveslight passing through the specimen. If an iris is included in opticalsystem 220, it would be placed at the back focal plane of 222. Theoptional iris receives light propagated by the collection element 222and passes light incident on its pupil area. Light incident on the areaof the iris around the pupil area is substantially blocked. The focusingelement 224 receives the light passed by the iris and focuses the lightto the light detector 232. The light detector 232 is configured toreceive light focused by the focusing element 224 and acquire anintensity image of the sample over each exposure time when the sample ison the specimen surface and illuminated by laser light from the angledirection device 212. The light detector 232 is configured to capture asequence of raw intensity images while the angle direction device 212provides laser light sequentially from different illumination angles atthe different sampling times.

During the high-resolution reconstruction phase, one or more processorsof the LFP system 200 interpret and process the raw image data from thesequence of images acquired during the image acquisition phase togenerate processed image data. The one or more processors interpret rawimage data from the sequence of acquired intensity images, transform therelatively low-resolution raw image data frames into Fourier space,iteratively update the transformed raw image data in Fourier space toreconstruct amplitude and phase data for a single high-resolution imageof the sample and the associated pupil function of the imaging system.In some cases, the one or more processors uses a differential phasecontrast (DPC) deconvolution procedure to acquire the quantitative phaseof the sample and use the phase data to correct for noise in thecaptured images such as those caused by speckles that may be caused bylaser light illumination. During the optional display phase, displaydata is sent to a display such as display 152 in FIG. 1 to display datasuch as the high-resolution image and other data.

C. LFP Systems with a Fiber Array Structure

Certain implementations of an LFP imaging system include an angulardirection device with a fiber array dome or other shaped structure forreceiving an array of optical fibers at fixed locations. In theseimplementations, the surface wall of the fiber array dome or otherstructure has an array of optical ports for receiving and holding inplace an array of optical fibers in directions that will provide laserillumination from a plurality of illumination angles. For example, theoptical ports may be apertures or other holder, each designed to receivea fiber connector at one end of an optical fiber or the one end of thefiber directly without the fiber connector. The other end of each of theoptical fibers is optically coupled to an optical switch. The opticalswitch is connected to another optical switch or directly to the lasersource(s). The optical switch(es) may be controlled by a controller orother computing device. In certain implementations, an optical switchmay couple one fiber channel to the laser light source at a time,providing illumination on the specimen surface plane at a certain angle.Although the surface wall is a dome-shape in the illustrated examplebelow, it would be understood that other shapes can be used. In oneembodiment, the surface of the fiber array structure may be a flat platewith slanted apertures. In another embodiment, the fiber array structuremay be a truss structure.

As mentioned above, the LFP imaging systems with a fiber array dome, oneor more optical switches can be used. In implementations where a singleoptical switch is used, the optical switch is configured to switch toeach of the optical fibers. In other cases, multiple optical switchesare used. For example, a cascade arrangement of multiple opticalswitches in series can be used. Each output end of an optical switch maybe connected to the input end of a separate optical switch to increasethe number of optical fiber outputs from the laser source(s) availablefor the fiber array dome design. An example of an optical switch thatcan be used in series is a mol 1×16 19″ 2 HU from Leoni Fiber Optics®that can switch the light output to 16 optical fibers given one inputlight source.

FIG. 3 is a schematic drawing of a cutaway view of an LFP imaging system300 comprising an angle direction device 312 having a fiber array dome314, in accordance with some implementations. The fiber array dome 314is configured to receive and direct optical fibers 301(1) through301(37) at a plurality of illumination angles to the sample plane. Thefiber array dome 314 comprises thirty-seven (37) optical ports 305(1)through 305(37) for receiving and holding optical fibers 301(1) through301 (37) with respective fiber connectors 303(1) through 303 (37). Inthis cutaway view, seven (7) optical ports 305(1) through 305(7) areshown with fiber connectors 303(1) through 303 (37) and their respectiveoptical fibers 301(1) through 301 (37). It would be understood that thefiber array dome 314 may be configured with more or fewer optical ports305. For example, the fiber array dome 314 could be configured withninety-seven (97) optical ports 305.

The LFP imaging system 300 further includes an optical switch 318. Eachoptical fiber 301 is shown with a first end located within a respectivefiber connector 303 held within a respective fiber port 305. The secondend of each optical fiber 301 is optically coupled to an optical switch318. Each optical port 305 holds the respective optical fiber 301 in aposition/direction such that when the laser light source(s) (not shown)is activated and the optical switch 318 is switched to provide laserlight to the respective optical fiber 301 at its second end, laser lightis propagated through the optical fiber 301 and directed out the firstend at a particular illumination angle. In the illustrated example, therespective fiber port 305(5) holds the respective optical fiber 301(5)in a position/direction to direct the laser light 304 spreadingconically from the first end of optical fiber 301(5) proximal of theoptical port 305(5). A bisecting arrow through the cone is showndepicting the illumination angle of the laser light from the first endof the optical fiber 301(5). A transparent layer 307 (e.g., slide) witha specimen surface 308 is also shown. The specimen surface 308 canreceive a sample (not shown) to be imaged. The transparent layer 307,the optical fibers 301, the optical ports 305, fiber connectors 303,and/or the optical switch 318, may be components of or may be separatecomponents from the LFP imaging system 300.

In the illustrated example, the LFP imaging system 300 further comprisesan optical system 320 having a collection element 322 and a focusingelement 324, and a light detector 332 for receiving light propagated bythe optical system 320. The collection element 322 (e.g., lens) has afocal length, f₁, and the focusing element 324 (e.g., lens) has a focallength, f₂. The collection element has an objective NA and is configuredto receive light issuing from a specimen when it is located on thespecimen surface 308. The focusing element 324 is configured to focuslight propagated from the collection element 322 to the light detector332. The sequence of illumination angles and the objective NA correspondto overlapping regions in the Fourier domain, as described in furtherdetail below. In the illustration, the optical system 320 is in a 4farrangement where the collection element 322 is located so that thesample plane is f₁ apart from it. The sample's Fourier plane is locatedf₁ away on the other side of 322. Fourier plane of the sample at thesample plane at specimen surface 308 is a focal length, f₂, away fromthe focusing element 324 and the focusing element 324 is located adistance of a focal length, f₂, away from the light detector. Otherarrangements can be used. For example, a 6f arrangement can be used. Asanother example, other optical elements (e.g., mirrors) can beintroduced into the optical path for other arrangements.

The LFP imaging system 300 further comprises a light detector 332 witheight discrete light elements. Although shown with eight discrete lightelements, it would be understood that other numbers of light elementscould be used. The collection element 322 receives light issuing fromthe sample (not shown) on the specimen surface 308 and the focusingelement 324 focuses light propagated from the collection element 322 tothe light detector 332.

In one embodiment, the LFP imaging system 300 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

While laser light 304 issuing from the optical fiber 301(5) is depictedas spreading conically outwards with a particular width, the width ofthis cone depends on the NA of the optical fiber 301(5). Different typesof optical fibers may be used to achieve desired illumination. By way ofexample, a single mode fiber optic with an NA in the range of 0.1-0.4may be used to achieve a narrower cone or a multi-mode fiber with an NAin the range of 0.1 to 0.8 may be used to achieve a wider cone.

The LFP system 300 also includes an optical switch 318 in opticalcommunication with the laser light source (not shown). The opticalswitch 318 is also in optically communication with optical fibers 301(1)through 301(37). The optical fibers 301(1) through 301(37) are coupledto respective fiber connectors 305(1) through 305(37). As introducedabove, the optical ports 305(1) through 305(37) are designed to directlaser light from the optical fibers 301(1) through 301(37) within thefiber connectors 305(1) through 305(37) to the specimen surface 308 suchthat a specimen on the specimen surface 308 may be sequentiallyilluminated with the laser light from thirty-seven (37) differentillumination angles during the image acquisition phase. During thisimage acquisition phase, the optical switch 318 switches to thedifferent optical fibers 301(1) through 301(37). By way of illustration,at the sampling time shown in FIG. 3, the optical switch 318 is switchedto provide laser light to optical fiber 301(5) within fiber connector303(5) located within optical port 303(5) to illuminate at theillumination angle depicted by the arrow. Once the sample has beenilluminated at a particular illumination angle, e.g., for at least theexposure time, the optical switch 318 switches to a different opticalfiber 301 to illuminate at a different illumination angle. This processof illumination and switching may continue until the sample has beenilluminated at each of illumination angles at each of the differentsampling times. The laser light source may be activated during theentire process or may be switched on at each exposure time during theprocess.

In FIG. 3, the optical ports 305(1) through 305(37) are shown ascircular apertures. It would be understood that the optical ports haveother shapes in other embodiments.

As shown in FIG. 3, each optical port 305 is configured along a fiberarray dome 314 to position the respective optical fiber 301 in aparticular direction. In order to approximate uniform illumination ateach illumination angle, the fiber array dome 314 can be designed sothat there is a uniform distance between each optical port 305 and thesample plane. In one embodiment, the fiber array dome 314 is hemisphereshaped with a constant radius so that there is a constant distancebetween each optical port 305 and the center of the circular surface atthe surface of the hemisphere. The circular surface at the surface ofthe hemisphere is kept near the outer surface of the transparent layer307 to approximate uniform illumination of the sample plane.

In certain implementations, the spacing between adjacent optical ports305 and the direction of the optical ports 305 are configured so thatthe laser light directed by the optical ports 305 generates adjacentillumination angles with overlapping regions in Fourier space of atleast a minimum amount (e.g. 60%, 65%, 70%, 80%, or 90%). The spacingbetween adjacent optical ports 305 and the size the fiber array dome 314may vary across implementations. In one embodiment, the spacing betweenadjacent optical ports 305 may be uniform. In one particularimplementation, the radius of the dome surface of the fiber array dome314 along which each optical port 305 is positioned is approximately 8mm. In this implementation, each optical port 303(i) may be spaced 1-5cm apart. A closer spacing, e.g. 1 cm, and a wider radius may be used ifthe collection element 322 of the optical system 320, as discussedfurther below, has a relative low NA for wide field-of-view imaging. Onthe other hand, a larger spacing, e.g. 5 cm, may be used if thecollection element 322 of the optical system 320 has a higher NA.

The optical switch 318 and/or other components of LFP system 300 may becontrolled by control signals from a processor(s) (e.g. process of thecontroller 140 of FIG. 1). The processor(s) retrieves instructionsstored in the CRM (e.g. internal memory 160 of FIG. 1) for performingvarious functions of the LFP system 300. For example, the processor(s)may be coupled with the optical switch 318 to send control signals toswitch the laser light from the laser source(s) to a particular opticalfiber 301 at a particular sampling time to direct the laser beam at oneof the illumination angles. In some cases, the controller may alsoactivate the laser light source(s). Alternatively, the optical switch318 may have an internal memory and processor and may be programmed tocause the optical switch 318 to switch between optical fibers 301 atpre-determined times.

While the optical switch 318 is described as a single optical switch, anarray of optical switches may be used in another implementation. In thiscase, an array of optical switches is connected in a cascading series toachieve N outputs. For example, each of the 16 outputs of a 1×16 19″ 2HU from Leoni Fiber Optics® may be connected with an input of another1×16 19″ 2 HU from Leoni Fiber Optics®. In such an arrangement, 256outputs could be achieved. While such a series may be extendedindefinitely (e.g. the output of one switch may be connected with theinput of another switch, which may be connected with the input of yetanother switch, etc.), each interface between switches introduces acertain amount of insertion loss/reflection. Accordingly, such a seriesof switches will cause some decrease in intensity of the laser lightfrom the laser light source. Thus, in this scenario, the intensity ofthe laser light source should be adjusted accordingly. This insertionloss may be avoided by using a single optical switch with theappropriate number of outputs.

At the instant in time illustrated in FIG. 3 angle direction device 312illuminates the specimen on specimen surface 308. The light incident onthe specimen is scattered by the physical features of the specimen as itpasses through the specimen. The collection element 322, which has aparticular objective NA, receives light passing through the specimen. Ifan iris is included in optical system 320, it would be placed at theback focal plane of collection element 322. The iris receives lightpropagated by the collection element 322 and passes light incident onits pupil area. Light incident on the area of the iris around the pupilarea is substantially blocked. The focusing element 324 receives thelight passed by the iris and focuses the light to the light detector332. The light detector 332 is configured to receive light focused bythe focusing element 324 and acquire a sequence of intensity images ofthe sample when the sample is on the specimen surface 308 and while theoptical switch 318 switches laser light between the different opticalfibers 301 at the different sampling times.

During the high-resolution reconstruction phase, one or more processorsof the LFP system 300 interpret and process the raw image data from thesequence of images acquired during the image acquisition phase togenerate processed image data. The one or more processors interpret rawimage data from the sequence of acquired intensity images, transform therelatively low-resolution raw image data frames into Fourier space,iteratively update the transformed raw image data in Fourier space toreconstruct amplitude and phase data for a single high-resolution imageof the sample and the associated pupil function of the imaging system.In some cases, the one or more processors uses a differential phasecontrast (DPC) deconvolution procedure to acquire the quantitative phaseof the sample and use the phase data to correct for noise in thecaptured images such as those caused by speckles that may be caused bylaser light illumination. During the optional display phase, displaydata is sent to a display such as display 152 in FIG. 1 to display datasuch as the high-resolution image and other data.

D. LFP Systems with a Rotatable Mirror and a Mirror Array

Certain implementations of an LFP imaging system include an angulardirection device with a two-axis rotatable mirror and a mirror array(e.g., faceted bowl shaped mirror array). In these implementations, acollimated laser beam is directed to the reflective surface of thetwo-axis rotatable mirror system such as Galvo mirror. The two-axisrotatable mirror system rotates its one or more lenses to receive andreflect the laser beam to each of a plurality of mirror elements in themirror array. The laser beam is reflected by each of the mirror elementsto direct the laser beam at the illumination angles to the sample plane.Changing the tilt angle of the two-axis rotatable mirror system allowsthe laser beam to be incident on different mirror elements and providesfor angularly varying illumination of the sample plane during the rawimage acquisition process.

FIG. 4 shows a schematic drawing of a cutaway view of an LFP imagingsystem imaging system 400 comprising an angle direction device 412 witha two-axis rotatable mirror 413 and a mirror array 414, in accordancewith some implementations. In FIG. 4, a laser light source (notdepicted) is activated and providing collimated laser light 404 to atwo-axis rotatable mirror 413 of the angle direction device 412. Atransparent layer 407 (e.g., slide) with a specimen surface 408generally at the sample plane is also shown. The specimen surface 408can receive a sample (not shown) to be imaged. One or more components ofthe angle direction device 412 may be separate from the LFP imagingsystem 400. The LFP imaging system 400 further comprises an opticalsystem 420 and a light detector 432 for receiving light propagated bythe optical system 420.

The LFP Imaging system 400 also includes an optical system 420 having acollection element 422 (e.g., lens) having a focal length, f₁, and afocusing element 424 (e.g., lens) having a focal length, f₂. Thecollection element has an objective NA and is configured to receivelight issuing from a specimen when it is located on the specimen surface407. The focusing element 424 is configured to focus light propagatedfrom the collection element 422 to the light detector 432. The sequenceof illumination angles and the objective NA correspond to overlappingregions in the Fourier domain, as described in further detail below. Inthe illustration, the optical system 420 is in a 4f arrangement wherethe collection element 422 is located so that the sample plane is f1apart from it. The sample's Fourier plane is located f1 away on theother side of 422. Fourier plane of the sample at the sample plane atspecimen surface 408 is a focal length, f₂, away from the focusingelement 424 and the focusing element 424 is located a distance of afocal length, f₂, away from the light detector. 408 Other arrangementscan be used. For example, a 6f arrangement can be used. As anotherexample, other optical elements (e.g., mirrors) can be introduced intothe optical path for other arrangements.

Angle direction device 412 includes a rotatable mirror 413 and a mirrorarray 414 having N mirrors 415. Rotatable mirror 413 may be configuredor configurable to rotate along two axes to orient its reflectivesurface at different positions during the image acquisition phase inorder to reflect laser light from the laser light source to each of theN mirrors 415 of the mirror array 414 at different sampling times. Eachmirror 415 of the mirror array 414 is oriented such that it reflectslaser light received from the rotatable mirror 413 to a sample plane atthe specimen surface 408. During the image acquisition phase, therotatable mirror 413 rotates to the different positions, in some casesholding at each position for at least an exposure time, to reflect laserlight from the laser light source to different mirrors 415 of the mirrorarray 414 to reflect the laser light at the sequence of illuminationangles described above in the context of FIG. 1. As such, the rotatablemirror 413 rotates such that collimated laser light 404 is reflected bythe rotatable mirror 413 to the mirrors of the mirror array 414 toilluminate the specimen at different illumination angles at differentsampling times. At the instant in time shown in the illustrated in FIG.4, the rotatable mirror 413 is rotated to a particular position to causethe collimated laser light 404 to be directed to one mirror 415 of themirror array 414 from which the collimated laser light 404 is reflected,illuminating the specimen at one of the illumination angles. Once thecollimated laser light 404 has illuminated the specimen at theparticular illumination angle, typically for at least an exposure time,the light detector 432 measures an intensity image and the rotatablemirror 413 is rotated to another position to cause the collimated laserlight 404 to be directed to another mirror of the mirror array 414,illuminating the specimen from another one of the illumination anglesand the light detector measures another intensity image. This processmay continue until the specimen has been illuminated at each of theillumination angles at each of the different sampling times.

While a single two-axis rotatable mirror 413 is depicted in FIG. 4, twomirrors, each of which is rotatable about a single axis such as in aGalvo mirror system, may be used in another embodiment. An example oftwo mirrors that can be used in such an embodiments are the mirrors 613and 614 discussed in greater detail below in the context of FIGS. 6 and8.

The rotatable mirror 413 may be controlled in a variety of manners. Byway of example, a controller, e.g. controller 140 of FIG. 1, may becoupled with the rotatable mirror 413 of FIG. 4. The controller mayaccess instructions stored in a memory, e.g. internal memory 160 ofFIG. 1. The instructions may be executable by the controller to causethe rotatable mirror 413 of FIG. 4 to or rotate to any given positionsuch that the specimen is illuminated at each of the illumination anglesat each of the different sampling times, as described in the precedingparagraph. Alternatively, the rotatable mirror 413 may have an internalmemory and processor and may be programmed to cause the rotatable mirror413 to follow a pre-determined rotation path.

The rotatable mirror 413 and/or other components of LFP system 400 maybe controlled by a controller (e.g. controller 140 of FIG. 1) with aprocessor and a computer readable medium (CRM) in communication with theprocessor. The controller retrieves instructions stored in the CRM (e.g.internal memory 160 of FIG. 1) for performing various functions of theLFP system 400. For example, the controller may be coupled with therotatable mirror 413 to send control signals to rotate the rotatablemirror 413 at a particular sampling time to direct the laser beam at oneof the illumination angles. In some cases, the controller may alsoactivate the laser light source(s). In one case, the laser lightsource(s) are turned on during the entire image acquisition phase. Inanother case, the laser light source(s) are activated to be turned onduring at least the exposure time of each intensity image acquisition.Alternatively, the rotatable mirror 413 or other system components mayhave an internal memory and processor and may be programmed to causerotatable mirror 413 to rotate at pre-determined times.

Optical system 420 may have some of the same or similar components andoperate in the same or similar manner as optical systems 220 and 320 ofFIGS. 3 and 4. Like in FIGS. 3 and 4, when the specimen on specimensurface 408 of FIG. 4 is illuminated at each of illumination angles ateach of the different sampling times, some of the light illuminating thespecimen is scattered by the specimen and passes through an opticalsystem 420. Optical system 420 includes a collection element 422 and afocusing element 424.

In one embodiment, the LFP imaging system 300 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

At the instant in time illustrated in FIG. 4, angle direction device 412illuminates the specimen on specimen surface 408. The light incident onthe specimen is scattered by the physical features of the specimen as itpasses through the specimen. The collection element 422, which has aparticular objective NA, receives light issuing from the specimen beingilluminated. If an iris is included in optical system 420, it would beplaced at the backfocal plane of 422. The iris receives light propagatedby the collection element 422 and passes light incident on its pupilarea. Light incident on the area of the iris around the pupil area issubstantially blocked. The focusing element 424 receive the light passedby the iris and focuses the light to the light detector 432.

Like in FIGS. 2-3, a light detector 432 of FIG. 4 receives light focusedby the focusing element 424 and acquires a sequence of intensity imagesof the specimen when the specimen is illuminated sequentially by theplurality of illumination angles at the different sampling times.

During the high-resolution reconstruction phase, one or more processorsof the LFP system 400 interpret and process the raw image data from thesequence of images acquired during the image acquisition phase togenerate processed image data. The one or more processors interpret rawimage data from the sequence of acquired intensity images, transform therelatively low-resolution raw image data frames into Fourier space,iteratively update the transformed raw image data in Fourier space toreconstruct amplitude and phase data for a single high-resolution imageof the sample and the associated pupil function of the imaging system.In some cases, the one or more processors uses a differential phasecontrast (DPC) deconvolution procedure to acquire the quantitative phaseof the sample and use the phase data to correct for noise in thecaptured images such as those caused by speckles that may be caused bylaser light illumination. During the optional display phase, displaydata is sent to a display such as display 152 in FIG. 1 to display datasuch as the high-resolution image and other data.

E. LFP Systems with a Rotatable Mirror and Lenses

Certain implementations of an LFP imaging system include an angulardirection device with a rotatable mirror and lenses. In theseimplementations, a collimated laser beam is directed to the reflectivesurface of the two-axis rotatable mirror such as Galvo mirror. Thetwo-axis rotatable mirror is placed at the focal plane of a first lens.The two-axis rotatable mirror is rotated to reflect the laser beam atdifferent angles to the first lens. The laser beam reflected from thetwo-axis rotatable mirror is focused by the first lens on the firstlens' back-focal plane, which coincides with the focal plane of a secondlens. The laser beam exits the second lens as a collimated beam andshines on the sample plane at an illumination angle, which isproportional to the angle at which the laser beam is reflected off therotatable mirror. In this way, the rotatable mirror can rotate (tilt) todirect the laser light at different illumination angles to the sampleplane to illumination a sample to be imaged.

FIG. 5 shows a schematic drawing of a side view of an example of an LFPimaging system 500 comprising an angular direction device 512 with atwo-axis rotatable mirror 513 and a first lens 514 and a second lens515, in accordance with some implementations. In FIG. 5, a laser lightsource (not depicted) is activated and providing collimated laser light504 to a two-axis rotatable mirror 513 of the angle direction device512. A transparent layer 507 (e.g., slide) with a specimen surface 508is also shown. The specimen surface 508 can receive a sample (not shown)to be imaged. One or more components of the angle direction device 512may be separate from the LFP imaging system 500.

The LFP Imaging system 500 also includes an optical system 520 having acollection element 522 (e.g., lens) having a focal length, f₁, and afocusing element 524 (e.g., lens) having a focal length, f₂. Thecollection element has an objective NA and is configured to receivelight issuing from a specimen when it is located on the specimen surface507. The focusing element 524 is configured to focus light propagatedfrom the collection element 522 to the light detector 532. The sequenceof illumination angles and the objective NA correspond to overlappingregions in the Fourier domain, as described in further detail below. Inthe illustration, the optical system 520 is in a 4f arrangement wherethe collection element 522 is located so that the sample plane is f₁apart from it. The sample's Fourier plane is located f₁ away on theother side of collection element 522. Fourier plane of the sample at thesample plane at specimen surface 508 is a focal length, f₂, away fromthe focusing element 524 and the focusing element 524 is located adistance of a focal length, f₂, away from the light detector. Otherarrangements can be used. For example, a 6f arrangement can be used. Asanother example, other optical elements (e.g., mirrors) can beintroduced into the optical path for other arrangements.

Angle direction device 512 includes a rotatable mirror 513 and a lenssystem including a first lens 514 and a second lens 515. The rotatablemirror 513 propagates collimated laser light 504 to the specimen surface508 via the lens system including first and second lenses 514 and 515.During an image acquisition phase, the rotatable mirror 513 isconfigured to rotate along two axes to direct collimated laser light 504to the first lens 514 at a sequence of incidence angles. The first lens514 focuses light 504. The first and second lenses 514 and 515 arepositioned such that the focal plane 516 of the first lens 514 coincideswith the back focal plane of the second lens 515. As such, the light 504focused by the first lens 514 is unfocused by the second lens 515 backinto a collimated beam. The rotatable mirror 513 is configured to rotatesuch that collimated laser light 504 is refracted by the first andsecond lenses 514 and 515 to illuminate the specimen with a collimatedbeam at each illumination angle of a sequence of illumination angles, asdescribed above in the context of FIG. 1. By way of illustration, at theinstant in time shown in FIG. 5, the rotatable mirror 513 is rotated toa particular position to cause the collimated laser light 504 to berefracted by the lenses 514 and 515, illuminating the specimen at one ofthe illumination angles. While the collimated laser light 504illuminates the specimen at the particular illumination angle, e.g., forat least the exposure time, the light detector 532 measures an intensityimage. Once the intensity image is acquired, the rotatable mirror 513may be rotated to another position to acquire another intensity image.When the rotatable mirror 513 is in the other position it causes thecollimated laser light 504 to be refracted at a different angle by thelenses 514 and 515, illuminating the specimen at a different one of theillumination angles and the light detector 532 acquires anotherintensity image. This process may continue until the specimen has beenilluminated at each of illumination angles at each of the differentsampling times and the light detector 532 acquires a sequence ofintensity images.

In one embodiment, the LFP imaging system 500 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

In addition to mitigating aberrations from speckles in image due to theuse of laser light, other mitigation measures may be taken to accountfor other aberrations in the LFP systems according to certainembodiments. For example, mitigation measures may be taken into accountfor aberration introduced when collimated laser light 504 enters thelenses 514 and 515 in different places. For example, in someimplementations, the lenses 514 and 515 are both F-theta lenses ratherthan conventional spherical lenses to reduce spherical aberration.Examples of F-theta lenses are commercially available such as an F-thetalens produced by Thor Labs®.

The rotatable mirror 513 may be controlled in a variety of manners. Byway of example, a processor (e.g., processor of controller 140 ofFIG. 1) may be in communication with the rotatable mirror 513 of FIG. 5.The processor may access instructions stored in a memory, e.g. internalmemory 160 of FIG. 1. The instructions may be executable by theprocessor to cause the rotatable mirror 513 of FIG. 5 to rotate to anygiven position such that the specimen is illuminated at each of theillumination angles at each of the different sampling times.Alternatively, the rotatable mirror 513 may have an internal memory andprocessor and may be programmed to cause the rotatable mirror 513 tofollow a pre-determined rotation path.

Generally, the rotatable mirror 513 and/or other components of LFPsystem 500 may be controlled with control signals sent by a processor(s)(e.g. processor of controller 140 of FIG. 1). The processor retrievesinstructions stored in a computer readable medium (CRM) (e.g. internalmemory 160 of FIG. 1) for performing various functions of the LFP system500. For example, the controller may be coupled with the rotatablemirror 513 to send control signals to rotate the rotatable mirror 513 ata particular sampling time to direct the laser beam at one of theillumination angles. In some cases, the controller may also activate thelaser light source(s). In one case, the laser light source(s) are turnedon during the entire image acquisition phase. In another case, the laserlight source(s) are activated to be turned on during at least theexposure time of each intensity image acquisition. Alternatively, therotatable mirror 513 may have internal memory and processor and may beprogrammed to cause rotatable mirror 513 to rotate at pre-determinedtimes and/or other system components may have internal memory andprocessor and may be programmed in its operations.

While a single two-axis rotatable mirror 513 is depicted in FIG. 5, twomirrors, each of which is rotatable about a single axis, may be used inanother embodiment. An example of two mirrors that can be used in suchan embodiments are the mirrors 613 and 614 and 813 and 814 discussed ingreater detail below in the context of FIGS. 6 and 8.

Optical system 520 may have the same or similar elements and operate inthe same or similar manner as optical systems 220, 320 and 420 of FIGS.3, 4, and 5. Like in FIGS. 3, 4, and 5, when the specimen on specimensurface 508 of FIG. 5 is illuminated at each of illumination angles ateach of the different sampling times, some of the light illuminating thespecimen is scattered by the specimen and passes through an opticalsystem 520. Optical system 520 includes a collection element 522 and afocusing element 524.

At the instant in time illustrated in FIG. 5 angle direction device 512illuminates the specimen on specimen surface 508. The light incident onthe specimen is scattered by the physical features of the specimen as itpasses through the specimen. The collection element 522, which has aparticular objective NA, receive light passing through the specimen. Ifan iris is included in optical system 520, it would be placed at theback focal plane of collection element 522. The iris receives lightpropagated by the collection element 522 and passes light incident onits pupil area. Light incident on the area of the iris around the pupilarea is substantially blocked. The focusing element 524 receives thelight passed by the iris and focuses the light to the light detector532.

Like in FIGS. 2-4, a light detector 532 of FIG. 5 receives light focusedby the focusing element 524 and acquires a sequence of intensity imagesof the specimen when the specimen is illuminated sequentially by theplurality of illumination angles at the different sampling times.

During the high-resolution reconstruction phase, one or more processorsof the LFP system 500 interpret and process the raw image data from thesequence of images acquired during the image acquisition phase togenerate processed image data. The one or more processors interpret rawimage data from the sequence of acquired intensity images, transform therelatively low-resolution raw image data frames into Fourier space,iteratively update the transformed raw image data in Fourier space toreconstruct amplitude and phase data for a single high-resolution imageof the sample and the associated pupil function of the imaging system.In some cases, the one or more processors uses a differential phasecontrast (DPC) deconvolution procedure to acquire the quantitative phaseof the sample and use the phase data to correct for noise in thecaptured images such as those caused by speckles that may be caused bylaser light illumination. During the optional display phase, displaydata is sent to a display such as display 152 in FIG. 1 to display datasuch as the high-resolution image and other data.

F. LFP Systems with a Circular Mirror Array on Flat Surface

Certain implementations of an LFP imaging system include an angulardirection device with a two-axis rotatable mirror system such as a Galvomirror system and a circular array of mirrors (mirror array) arranged ona flat surface. For example, the mirror array may be an arrangement ofmirrors in concentric circles along a flat surface. In theseimplementations, a collimated laser beam is directed to the reflectivesurface of the two-axis rotatable mirror. The two-axis rotatable mirrorsystem rotates its one or more mirrors to receive and reflect the laserbeam to each of a plurality of mirror elements in the mirror array. Thelaser beam is reflected by each of the mirror elements to direct thelaser beam at the illumination angles to the sample plane. Changing thetilt angle of the two-axis rotatable mirror system allows the laser beamto be incident on different mirror elements and provides for angularlyvarying illumination of the sample plane during the raw imageacquisition process.

FIG. 6 shows a schematic drawing of an orthogonal view of an LFP imagingsystem imaging system 600 with a circular mirror array 615 on a flatsurface, in accordance with some implementations. A transparent layer607 (e.g., slide) with a specimen surface 608 generally at the sampleplane is also shown. The specimen surface 608 can receive a sample (notshown) to be imaged.

The LFP imaging system imaging system 600 comprises an angle directiondevice 612 with a first rotatable mirror 613 and second rotatable mirror614, and a circular mirror array 615 having ninety five (95) mirrors616(1)-616(95). More or fewer mirrors 616 may be used. One or morecomponents of the angle direction device 612 may be separate from theLFP imaging system 600. The LFP imaging system 600 further comprises anoptical system 620 and an image sensor system 630 with one or more lightdetectors for receiving light propagated by the optical system 620.

The optical system 620 comprises a collection element 622 (e.g., lens)having a focal length, f₁, and a focusing element 624 (e.g., lens)having a focal length, f₂. The collection element has an objective NAand is configured to receive light issuing from a specimen when it islocated on the specimen surface. The focusing element 624 is configuredto focus light propagated from the collection element 622 to the lightdetector of the image sensor system 630. The sequence of illuminationangles and the objective NA correspond to overlapping regions in theFourier domain, as described in further detail below. In theillustration, the optical system 620 is in a 4f arrangement where thecollection element 622 is located so that the sample plane is f1 apartfrom it. The sample's Fourier plane is located f1 away on the other sideof 622. Fourier plane of the sample at the sample plane at specimensurface 608 is a focal length, f₂, away from the focusing element 624and the focusing element 624 is located a distance of a focal length,f₂, away from the light detector. 608 Other arrangements can be used.For example, a 6f arrangement can be used. As another example, otheroptical elements (e.g., mirrors) can be introduced into the optical pathfor other arrangements.

Angle direction device 612 includes first and second rotatable mirrors613 and 614 and a circular mirror array 615 having ninety five (95)mirrors 616(1)-616(95). First and second rotatable mirrors 613 and 614may rotate each rotate about a different axis such that they areconfigured to be rotated to reflect laser light from the laser lightsource to each of the ninety five (95) mirrors 616(1)-616(95). The firstrotatable mirror 613 is configured to reflect collimated laser light 604from the laser light source (not shown) to the second rotatable mirror614. The second rotatable mirror 614 is configured to reflect to reflectthe collimated laser light 604 from the first rotatable mirror 613 toone of the mirrors 616(1)-616(95). Each of the mirror 616(1)-616(95) inthe mirror array 615 may be oriented such that it reflects laser lightreceived from the second rotatable mirrors 614 to the sample plane atone of the illumination angles in a sequence of illumination angles, asdescribed above in the context of FIG. 1. As such, the first and secondrotatable mirrors 613 and 614 are configured to rotate such thatcollimated laser light 604 may be reflected by the first and secondrotatable mirrors 613 and 614 and directed to the mirrors 616(1)-616(95)to illuminate the specimen at each illumination angles in the sequenceof illumination angles. By way of illustration, at a particular samplingtime, the first and second rotatable mirrors 613 and 614 may be rotatedto particular orientations to cause the collimated laser light 604 to bedirected to a particular mirror 616(i) of the circular mirror array 615from which the collimated laser light 604 is reflected, illuminating thespecimen at a particular one of the illumination angles. While thecollimated laser light 604 illuminates the sample plane at a particularillumination angle, e.g., for at least an exposure time, a lightdetector(s) of the image sensor system 630 acquires an intensity imageand then the rotatable mirrors 613 and 614 may be rotated to cause thecollimated laser light 604 to be directed to another mirror 616(i+1),illuminating the specimen from another one of the illumination angles sothat the light detector may generate another intensity image. Thisprocess may continue until the specimen has been illuminated at each ofillumination angles at each of the different sampling times.

At the instant in time depicted in FIG. 6, a laser light source (notdepicted) is activated and providing collimated laser light 604 to thefirst rotatable mirror 613 of the angle direction device 612. The firstrotatable mirror 613 is oriented to reflect collimated laser light 604to the second rotatable mirror 614 which reflects the collimated laserlight 604 to a particular mirror 616(i) of the circular mirror array 615from which the collimated laser light 604 is reflected to the specimenplane which is approximately at the specimen surface 608.

In one embodiment, the LFP imaging system 600 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

Although the mirror array 615 is illustrated with ninety five (95)mirrors 616, other numbers of mirrors can be used in the mirror array615. Each mirror 616(i) of the mirror array 615 is coupled at one end ofa rectangular tower coupled to a plate. In one embodiment, therectangular towers are 3D-printed structures. Each tower's top surfaceis sloped at a certain angle such that collimated laser light 604 may bereflected towards the sample plane at one of the illumination angles inthe sequence of illumination angles described above.

FIG. 7A is a plan view of a mirror array 715 with a similarconfiguration the mirror array 615 shown in FIG. 6, according to anembodiment. The mirror array 715 has a diameter, d. FIG. 7B is anillustration of the Fourier spectrum region 750 covered by the angularvarying illumination of the mirror array 715 shown in FIG. 7A. As shownby FIG. 7B, the mirrors 616(1)-616(95) are arranged to provideillumination to the sample plane such that contiguous regions produce60% overlap of the specimen's spectrum in the Fourier domain, as shownin FIG. 7B. As discussed above, other embodiments may have a greateroverlap, for example, at least 70%, at least 80%, etc.

In some implementations, a neutral density filter is placed on each ofthe mirrors 616(1)-616(95) in the mirror array 615. Use of such neutraldensity filters allows for increasing the input laser intensity toobtain higher SNR in dark field images while preventing the bright fieldimages from over-exposure.

The first and second rotatable mirrors 613 and 614 may be controlled ina variety of manners. By way of example, a controller, e.g. controller140 of FIG. 1, may be coupled with the first and second rotatablemirrors 613 and 614 of FIG. 6. The controller may access instructionsstored in a memory, e.g. internal memory 160 of FIG. 1. The instructionsmay be executable by the controller to cause the first and secondrotatable mirrors 613 and 614 of FIG. 6 to or rotate to any givenposition such that the specimen is illuminated at each of theillumination angles at each of the different sampling times, asdescribed in the preceding paragraph. Alternatively, the first andsecond rotatable mirrors 613 and 614 may have an internal memory andprocessor and may be programmed to cause each of the first and secondrotatable mirrors 613 and 614 to follow a pre-determined rotation path.

The first and second rotatable mirrors 613 and 614 and/or othercomponents of LFP system 600 may be controlled by control signals from aprocessor(s) (e.g., a processor of the controller 140 of FIG. 1). Theprocessor(s) retrieves instructions stored in a computer readable medium(CRM) (e.g. internal memory 160 of FIG. 1) for performing variousfunctions of the LFP system 400. For example, the processor(s) may becoupled with the first and second rotatable mirrors 613 and 614 to sendcontrol signals to rotate the first and second rotatable mirrors 613 and614 at particular sampling times to direct the laser beam to thedifferent illumination angles. In some cases, the processor(s) may alsosend control signals to activate the laser light source(s). In one case,the laser light source(s) are turned on during the entire imageacquisition phase. In another case, the laser light source(s) areactivated to be turned on during at least the exposure time of eachintensity image acquisition. Alternatively, the first and secondrotatable mirrors 613 and 614 may have an internal memory and processorand may be programmed to cause first and second rotatable mirrors 613and 614 to rotate at pre-determined times. Also, other system componentsmay have an internal memory and a processor and may be programmed in itsoperations.

While two rotatable mirrors 613 and 614 are depicted in FIG. 6, a singlemirror rotatable about two different axes can be used in anotherembodiment. Such a configuration of a single mirror is discussed ingreater detail above in the context of FIGS. 4 and 5.

Optical system 620 may have the same or similar elements and operate inthe same or similar manner as optical systems 220, 320, 420 and 520 ofFIGS. 3, 4, 5 and 6. Like in FIGS. 3, 4, 5 and 6, when the specimen onspecimen surface 605 of FIG. 6 is illuminated at each of illuminationangles at each of the different sampling times, some of the lightilluminating the specimen is scattered by the specimen and passesthrough an optical system 620. Optical system 620 includes a collectionelement 622 and a focusing element 624.

At the instant in time illustrated in FIG. 6, angle direction device 602illuminates the specimen on specimen surface 605. The light incident onthe specimen is scattered by the physical features of the specimen as itpasses through the specimen. The collection element 622, which has aparticular objective NA, receives light passing through the specimen. Ifan iris is included in optical system 620, it would be placed at theback focal plane of collection element 622. The iris receives lightpropagated by the collection element 622 and passes light incident onits pupil area. Light incident on the area of the iris around the pupilarea is substantially blocked. The focusing element 624 receives thelight passed by the iris and focuses the light to the light detector630.

A light detector of the image sensor system 630 of FIG. 6 receives lightfocused by the focusing element 624 and acquires an intensity image ofthe specimen when the specimen is illuminated at each of theillumination angles at each of the different sampling times. During thehigh-resolution reconstruction phase, one or more processors of the LFPsystem 600 interpret and process the raw image data from the sequence ofimages acquired during the image acquisition phase to generate processedimage data. The one or more processors interpret raw image data from thesequence of acquired intensity images, transform the relativelylow-resolution raw image data frames into Fourier space, iterativelyupdate the transformed raw image data in Fourier space to reconstructamplitude and phase data for a single high-resolution image of thesample and the associated pupil function of the imaging system. In somecases, the one or more processors uses a differential phase contrast(DPC) deconvolution procedure to acquire the quantitative phase of thesample and use the phase data to correct for noise in the capturedimages such as those caused by speckles that may be caused by laserlight illumination. During the optional display phase, display data issent to a display such as display 152 in FIG. 1 to display data such asthe high-resolution image and other data.

1) Example of an LFP Imaging System with Circular Mirror Array on a FlatSurface

The dimensions and/or types of components of LFP Imaging systemsdisclosed herein may vary across implementations depending on thedesired resolution of reconstructed images generated by the system, thefield of view of view and/or NA of optical systems used in the LFPImaging system, types of specimens being imaged by the LFP Imagingsystem, and other system parameters.

One particular implementation of an LFP Imaging system with a circulararray on a flat surface has components similar to those described withrespect to FIG. 6 and FIG. 7A. In this particular implementation, theoptical system is a 4f system with collection element being a 0.1 NAOlympus® 4× objective lens and focusing element being a Thorlabs®200-mm-focal-length tube lens. The light detector is a PCO.edge® 5.5 16bit sCMOS sensor. The sensor has a pixel size of 6.5 mm and a maximumframe-rate of 100 Hz at 1920×1080 pixel resolution for a global shuttermode. In this implementation, the sensor size limits the available fieldof view of a specimen to be 2.7 mm by 1.5 mm. In this implementation,the laser light source is a 457 nm 1 W laser beam, which ispinhole-filtered and collimated, using standard collimation techniques.After collimation, the collimated laser light is 1 cm in diameter andhas a 150 mW in power. Since the beam diameter the collimated laserlight 1 cm, the collimated laser light covers the entire field of viewcaptured by the light detector (2.7 mm by 1.5 mm after magnification).

In this particular implementation, the circular mirror array of mirrorshas a height h of 30 cm. The mirrors are placed 40 cm away from theplane of the specimen surface. Rotatable mirror, which is a 2D Galvomirror device (GVS 212), guides collimated laser light such that thecentral part of its Gaussian profile (approximately 40% of total outputarea) is incident on the input of the rotatable mirror, which is also 2DGalvo mirror device (GVS 212). Rotatable mirror then guides collimatedlaser light to individual mirrors. Each mirror is a 19 mm×19 mm firstsurface mirror attached to a 3D-printed rectangular tower. Each tower'stop surface is sloped at a certain angle such that collimated laserlight may be reflected towards the specimen surface at one of theillumination angles in the sequence of illumination angles describedabove.

The mirrors of the mirror array are arranged to provide illumination toa sample plane such that contiguous elements produce 60% overlap of thespecimen's spectrum in the Fourier domain. In this implementation, thetotal illumination (NA_(ti)) is 0.325 with the resulting system NA(NA_(sys)) being NA_(obj)+NA_(ti)=0.1+0.325=0.425. In this particularimplementation, the image reconstruction effectively increases the NA ofthe optical system by a factor of 4.25.

In this particular implementation, to achieve the maximum frame rate ofthe sCMOS sensor in light detector, the exposure time is set to itsminimum, at 500 microseconds. The light detector and rotatable mirrorsand are externally triggered every 10 milliseconds, resulting in 0.96seconds of total capturing time for 95 intensity images (1 for eachillumination angle) and 1 dark noise image.

G. LFP Systems with a Rectangular Mirror Array on Flat Surface

Certain implementations of an LFP imaging system include an angulardirection device with a two-axis rotatable mirror system such as a Galvomirror system and a rectangular array of mirrors (mirror array) arrangedon a flat surface. For example, the mirror array may be an arrangementof mirrors in a rectangular array along a flat surface. In theseimplementations, a collimated laser beam is directed to the reflectivesurface of the two-axis rotatable mirror. The two-axis rotatable mirrorsystem rotates its one or more lenses to receive and reflect the laserbeam to each of a plurality of mirror elements in the mirror array. Thelaser beam is reflected by each of the mirror elements to direct thelaser beam at the illumination angles to the sample plane. Changing thetilt angle of the two-axis rotatable mirror system allows the laser beamto be incident on different mirror elements and provides for angularlyvarying illumination of the sample plane during the raw imageacquisition process.

FIG. 8 shows a schematic drawing of an orthogonal view of an LFP imagingsystem imaging system 800 with a rectangular mirror array 815 on a flatsurface, in accordance with some implementations. The LFP imaging systemimaging system 800 comprises an angle direction device 812 with a firstrotatable mirror 813 and second rotatable mirror 814, and a rectangularmirror array 815 having a hundred (100) mirrors 804(1)-804(100). In FIG.8, a laser light source (not depicted) is activated and providingcollimated laser light 804 to an angle direction device 812. Atransparent layer 807 (e.g., slide) with a specimen surface generally atthe sample plane is also shown. The specimen surface can receive asample (not shown) to be imaged. One or more components of the angledirection device 812 may be separate from the LFP imaging system 800.The LFP imaging system 800 further comprises an optical system 820 andan image sensor system 830 with one or more light detectors forreceiving light propagated by the optical system 820.

The LFP Imaging system 800 also includes an optical system 820 having acollection element 822 (e.g., lens) having a focal length, f₁, and afocusing element 824 (e.g., lens) having a focal length, f₂. Thecollection element has an objective NA and is configured to receivelight issuing from a specimen when it is located on the specimensurface. The focusing element 824 is configured to focus lightpropagated from the collection element 822 to the light detector of theimage sensor system 830. The sequence of illumination angles and theobjective NA correspond to overlapping regions in the Fourier domain, asdescribed in further detail below. In the illustration, the opticalsystem 820 is in a 4f arrangement where the collection element 822 islocated so that the sample plane is f₁ apart from it. The sample'sFourier plane is located f₁ away on the other side of the collectionelement 822. Fourier plane of the sample at the sample plane at specimensurface 808 is a focal length, f₂, away from the focusing element 824and the focusing element 824 is located a distance of a focal length,f₂, away from the light detector. Other arrangements can be used. Forexample, a 6f arrangement can be used. As another example, other opticalelements (e.g., mirrors) can be introduced into the optical path forother arrangements.

The angle direction device 812 includes first and second rotatablemirrors 613 and 814 and a rectangular mirror array 815 having a hundred(100) mirrors 804(1)-804(100). First and second rotatable mirrors 813and 814 may rotate each rotate about a different axis such that they areconfigured to be rotated to reflect laser light from the laser lightsource to each of the hundred (100) mirrors 804(1)-804(100). By way ofillustration, the first rotatable mirror 813 may reflect collimatedlaser light 804 from the laser light source (not shown) to the secondrotatable mirror 814. The second rotatable mirror 814 may then reflectthe collimated laser light 804 from the first rotatable mirror 813 toany of the mirrors 816(1)-816(100). Each of the mirror 816(1)-816(100)in the mirror array 815 may be oriented such that it reflects laserlight received from the first and second rotatable mirrors 813 and 814to the sample plane at one of the illumination angles in a sequence ofillumination angles, as described above in the context of FIG. 1. Assuch, the first and second rotatable mirrors 813 and 814 may beconfigured to rotate such that collimated laser light 804 may bereflected by the first and second rotatable mirrors 813 and 814 anddirected to the mirrors 816(1)-816(100) to illuminate the specimen ateach illumination angles in the sequence of illumination angles. By wayof illustration, at a particular sampling time, the first and secondrotatable mirrors 813 and 814 may be rotated to a particular position tocause the collimated laser light 804 to be directed to a particularmirror 816(i) of the rectangular mirror array 815 from which thecollimated laser light 804 is reflected, illuminating the specimen at aparticular one of the illumination angles. Once the collimated laserlight 804 has illuminated the specimen at the particular illuminationangle, e.g., for at least an exposure time, a light detector of theimage sensor system 630 acquires an intensity image and the rotatablemirrors 813 and 814 may be rotated to another position to cause thecollimated laser light 804 to be directed to another mirror 816(i+1),illuminating the specimen from another one of the illumination angles sothat the light detector may generate another intensity image. Thisprocess may continue until the specimen has been illuminated at each ofillumination angles at each of the different sampling times.

In one embodiment, the LFP imaging system 800 also includes one or moreprocessors and computer readable medium (CRM) (e.g., memory) incommunication with the processor(s). The one or more processors andcomputer readable medium may be part of a controller such as thecontroller 140 described with respect to FIG. 1. Theprocessor-executable code (or “instructions”) for performing the LFPimaging method can be stored on the CRM or other memory. Theprocessor(s) and can retrieve the processor-executable code from the CRMor other memory to perform various functions or operations of the LFPimaging method. The CRM or other memory can store raw and/or processedimage data. In some implementations, the CRM or other memory canadditionally or alternatively include a volatile memory array fortemporarily storing code to be executed as well as image data to beprocessed, stored, or displayed.

Although the mirror array 815 is illustrated with one hundred (100)mirrors 816, other numbers of mirrors can be used in the mirror array815. Each mirror 816(i) of the mirror array 815 comprises a surfacemirror affixed at one end of a rectangular tower. In one embodiment, therectangular towers are 3D-printed structures. Each tower's top surfaceis sloped at a certain angle such that collimated laser light 804 may bereflected towards the sample plane at one of the illumination angles inthe sequence of illumination angles described above.

In some implementations, a neutral density filter is placed on each ofthe mirrors 816(1)-816(100) in the mirror array 815. Use of such neutraldensity filters allows for increasing the input laser intensity toobtain higher SNR in dark field images while preventing the bright fieldimages from over-exposure.

The first and second rotatable mirrors 813 and 814 may be controlled ina variety of manners. By way of example, a controller, e.g. controller140 of FIG. 1, may be coupled with the first and second rotatablemirrors 813 and 814 of FIG. 6. The controller may access instructionsstored in a memory, e.g. internal memory 160 of FIG. 1. The instructionsmay be executable by the controller to cause the first and secondrotatable mirrors 813 and 814 of FIG. 6 to or rotate to any givenposition such that the specimen is illuminated at each of theillumination angles at each of the different sampling times, asdescribed in the preceding paragraph. Alternatively, the first andsecond rotatable mirrors 813 and 814 may have an internal memory andprocessor and may be programmed to cause each of the first and secondrotatable mirrors 813 and 814 to follow a pre-determined rotation path.

The first and second rotatable mirrors 813 and 814 and/or othercomponents of LFP system 800 may be controlled by a controller (e.g.controller 140 of FIG. 1) with a processor and a computer readablemedium (CRM) in communication with the processor. The controllerretrieves instructions stored in the CRM (e.g. internal memory 160 ofFIG. 1) for performing various functions of the LFP system 400. Forexample, the controller may be coupled with the first and secondrotatable mirrors 813 and 814 to send control signals to rotate thefirst and second rotatable mirrors 813 and 814 at a particular samplingtime to direct the laser beam at one of the illumination angles. In somecases, the controller may also activate the laser light source(s). Inone case, the laser light source(s) are turned on during the entireimage acquisition phase. In another case, the laser light source(s) areactivated to be turned on during at least the exposure time of eachintensity image acquisition. Alternatively, the first and secondrotatable mirrors 813 and 814 or other system components may have aninternal memory and processor and may be programmed to cause first andsecond rotatable mirrors 813 and 814 to rotate at pre-determined times.

While two rotatable mirrors 813 and 814 are depicted in FIG. 6, a singlemirror rotatable about two different axes can be used in anotherembodiment. Such a configuration of a single mirror is discussed ingreater detail above in the context of FIGS. 4 and 5.

Optical system 820 may have the same or similar elements and operate inthe same or similar manner as optical systems 220, 320, 420 and 520 ofFIGS. 3, 4, 5 and 6. Like in FIGS. 3, 4, 5 and 6, when the specimen onspecimen surface 605 of FIG. 6 is illuminated at each of illuminationangles at each of the different sampling times, some of the lightilluminating the specimen is scattered by the specimen and passesthrough an optical system 820. Optical system 820 includes a collectionelement 822 and a focusing element 824.

At the instant in time illustrated in FIG. 6 angle direction device 802illuminates the specimen on specimen surface 805. The light incident onthe specimen is scattered by the physical features of the specimen as itpasses through the specimen. The collection element 822, which has aparticular objective NA, receives light passing through the specimen. Ifan iris is included in optical system 820, it would be placed at theback focal plane of 822. The iris receives light propagated by thecollection element 822 and passes light incident on its pupil area.Light incident on the area of the iris around the pupil area issubstantially blocked. The focusing element 824 receives the lightpassed by the iris and focuses the light to the light detector ofimaging system 830.

A light detector of the image sensor system 830 of FIG. 8 receives lightfocused by the focusing element 824 and acquires an intensity image ofthe specimen when the specimen is illuminated at each of theillumination angles at each of the different sampling times. During thehigh-resolution reconstruction phase, one or more processors of the LFPsystem 800 interpret and process the raw image data from the sequence ofimages acquired during the image acquisition phase to generate processedimage data. The one or more processors interpret raw image data from thesequence of acquired intensity images, transform the relativelylow-resolution raw image data frames into Fourier space, iterativelyupdate the transformed raw image data in Fourier space to reconstructamplitude and phase data for a single high-resolution image of thesample and the associated pupil function of the imaging system. In somecases, the one or more processors uses a differential phase contrast(DPC) deconvolution procedure to acquire the quantitative phase of thesample and use the phase data to correct for noise in the capturedimages such as those caused by speckles that may be caused by laserlight illumination. During the optional display phase, display data issent to a display such as display 152 in FIG. 1 to display data such asthe high-resolution image and other data.

As described above, mirror arrays in angular direction devices of LFPImaging systems can take a wide variety of shapes and sizes. By way ofexample, FIGS. 9A and B show examples of an 8×8 mirror array 900 and a6×6 mirror array 904, respectively, according to embodiments.

While FIGS. 5-9B show various examples of shapes and sizes of mirrorarrays in angular direction devices of LFP Imaging systems, such mirrorarrays are not limited to the bowl-shaped surface, flat concentriccircular ring surface, and/or flat rectangular surface arrangementsshown in FIGS. 5-9B, respectively. Rather, such mirror arrays may takeany symmetric or asymmetric round or polygonal shape, e.g. a rectangle,an octagon, an oval, an ellipse, etc.

II. LFP Imaging Methods

According to certain implementations, the LFP imaging method discussedin the context of FIG. 11 operates most effectively for a thin sample.The thinness of the sample depends on the bigger value between theillumination NA and the objective NA. In one case, the thinness can berelated to these value by pi/(kzmax) where kzmax is defined bymax(k0̂2−sqrt(k0̂2−(k0*NAobj)̂2), k0̂2−sqrt(k0̂2−(k0*NAillu)̂2)) which isdescribed in Ou, Xiaoze et al., “High numerical aperture Fourierptychography: principle, implementation and characterization,” OpticsExpress, p 3472-3492 (Feb. 9, 2015). For example, for NAillu=0.7 andNAobj=0.5, hmax will be 1.75*lambda, which corresponds to 822 nm forlambda=470 nm. The sample may be thicker than this in many cases.

For thin samples, the sample may be approximated as two dimensional,similar to a thin transparent film with a particular absorption andphase profile. In a general application of LFP methods to a specificimplementation using an optical system such as optical system 120 ofFIG. 1 (e.g. a 4f microscope), when a sample is on a specimen surface(e.g. on the stage of the 4f microscope) it may be perpendicularlyilluminated by a light source that is coherent both temporally (i.e.monochromatic) and spatially (i.e. plane wave). The light fieldscattered by features of the sample the sample is Fourier transformedwhen it passes through the collection optical element of the opticalsystem (e.g. objective lens microscope) and arrives at the back-focalplane of the collection optical element. The field is then Fouriertransformed again as it propagates through the focusing optical element(e.g. a microscope's tube lens) to be imaged onto a light detector (e.g.a camera sensor or in a microscopist's eyes). The amount of the sample'sdetail that the optical system can capture is defined by the NA(NA_(obj)) of the collection element, which physically limits the extentof the sample's Fourier spectrum being transmitted to the lightdetector. Thus, the NA_(obj) acts as a low-pass filter in the opticalsystem with a coherent illumination source.

For illustrative purposes, the following discussion is limited to a onedimensional case. The one dimensional case described below may bedirectly extended to two dimensions for a thin sample. Under theillumination of a same light source at an angle θ with respect to thesample's normal, the field at the sample plane, ψ_(oblique)(x), can bedescribed as:

ψ_(oblique)(x)=ψ_(sample)(x)exp(jk ₀ sin θ)  (Eqn. 1)

where ψ_(sample)(x) is the sample's complex spatial distribution, x is aone-dimensional spatial coordinate, and k₀ is given by 2π/λ where λ isthe illumination wavelength. This field is Fourier transformed by thecollection element, becoming:

ψ_(oblique)(k)=∫_(−∞) ^(∞) ψ_(sample)(x)exp(jk ₀ sin θ)exp(−jkx)dx=ψ_(sample)(k−k ₀ sin θ)  (Eqn. 2)

at the back-focal plane of the collection element, where ψ_(oblique) andψ_(sample) are the Fourier transforms of ψ_(oblique) and ψ_(sample),respectively, and k is a one dimensional coordinate in k-space.ψ_(sample)(k) is shown to be laterally shifted at the collectionelement's back-focal plane by k₀ sin θ. Because NA_(obj) is physicallyfixed, a different sub-region of ψ_(sample)(k) is relayed down theimaging system. Thus, more regions of ψ_(sample)(k) are acquirable bycapturing many intensity images under varying illumination angles thanwould be acquirable by only capturing a single image under particularillumination. Each sub-sampled Fourier spectrum from the collectionelement's back-focal plane is Fourier transformed again by the focusingoptical element, and the field's intensity value may captured by a lightdetector of the imaging system.

Due to the loss of phase information in the intensity measurement, thesub-sampled images cannot be directly combined in the Fourier domain. Assuch, an LFP procedure, e.g. the process described below in the contextof FIG. 11, may be used to reconstruct the phase and amplitude of theexpanded Fourier spectrum. As discussed above, an angle directionmechanism may illuminate a sample with laser light at a sequence ofillumination angles such that each the intensity images overlap inFourier space at least a certain minimum amount (e.g. 65% overlap, 75%overlap, 70% overlap, 80% overlap, in a range of 10%-65% overlap, in arange of 65%-75% overlap, etc.). This redundancy allows the LFPprocedure to be used to infer the missing phase information through aniterative method which is described below in the context of FIG. 11.

A. An Exemplary LFP Imaging Method

FIG. 10 is a flowchart of operations of an LFP imaging method 1000 thatcan be implemented by various LFP imaging systems described herein, inaccordance with certain embodiments. At 1010, a sample plane issuccessively illuminated at a sequence of N illumination angles using anangle direction device to direct laser light from a laser light source.During an image acquisition phase, the sample being imaged is located atthe sample plane typically on a specimen surface and the sample isilluminated at a sequence of N illumination angles using a laser lightsource. By way of example, as described above in the context of FIG. 6,the rotatable mirrors 613 and 614 are rotated to direct collimated laserlight 604 to different mirrors 816(1)-816(95) of the mirror array 615 atdifferent sampling times during the image acquisition phase. Thedifferent mirrors 816(1)-816(95) of the mirror array 615 reflect thecollimated laser light 604 at a sequence of N illumination angles atdifferent sample times. At each sampling time, the rotatable mirrors 613and 614 are positioned such that they direct the collimated laser light604 to one of mirrors 816(1)-816(95), illuminating the specimen at asequence of 95 illumination angles.

At 1020, N raw intensity images of the sample are acquired by the lightdetector(s). By way of example, the light detector(s) of the imagingsystem 630 in FIG. 6 may capture a sequence of ninety five (95)intensity images of the sample when it is on the specimen surface 608and while collimated laser light 604 is reflected by the ninety five(95) different mirrors 616(1)-616(95) at ninety five (95) illuminationangles to the sample plane at the specimen surface 608.

In one embodiment, the light detector may also capture an additional“dark field” intensity image of the sample when the sample is notilluminated.

At 1030, a high resolution image of the sample and/or pupil function isconstructed using an Embedded Pupil Function Recovery (EPRY) processwith one or more processors of the FLP imaging system. An example of anEPRY process is described below in detail with reference to FIG. 11. Atoptional operation 1040, a display may receive image data such as thehigher-resolution image data and/or other data, and display the data ona display (e.g., display 152 of FIG. 1). Then, the LFP method ends(1050) an imaging cycle.

FIG. 11 is a flowchart depicting operations of an exemplary EPRY process1100, according to embodiments. At operation 1110, the sample spectrumand pupil function are initialized as S₀(u) and P₀(u) respectively. Inaddition, the outer loop index variable, b, is set to 1 (firstiteration) and the inner loop index variable, a, is set to 0. Outer loopindex variable, b is the index incrementing the reconstruction processiterations and inner loop index variable, a, is the index incrementingthe incidence angle. In the cycles of the inner loop, N acquired rawintensity images are addressed in the sequence: I_(U) _(a) (r), a=0 toN−1, where N is the number of acquired images, and each is considered inturn, with both the pupil function and sample spectrum updated at eachloop.

In one embodiment, the initial sample spectrum S₀(u) may be determinedby first initializing a sample image in the spatial domain, and thenapplying a Fourier transform to obtain an initialized sample spectrum inthe Fourier domain. In some cases, the initial guess may be determinedas a random complex matrix (for both intensity and phase). In othercases, the initial guess may be determined as an interpolation of thelow-resolution intensity measurement with a random phase. An example ofan initial guess for S₀(u) may be interpolated from one of the capturedintensity images. Another example of an initial guess is a constantvalue. The Fourier transform of the initial guess can be a broadspectrum in the Fourier domain.

In one embodiment, the initial pupil function guess P₀(u) may be acircular shaped low-pass filter, with all ones inside the pass band,zeros out of the pass band and uniform zero phase. In one example, theradius of the pass band is NA×2π/λ, where NA is the numerical apertureof the filtering optical element (e.g., objective lens) and λ is theillumination wavelength. An example of an initial pupil function guesswould be based on assuming the system is aberration free, phase=0.

At operation 1112, it is determined whether b=1 i.e. it is the firstiteration of the outer loop. If it is determined that it is not thefirst iteration, then the initial pupil function and the sample spectrumin the Fourier domain are set to the data determined in the last cycleof the inner loop: S₀(u)=S_(M-1)(u) and P₀(u)=P_(M-1)(u) at operation1114.

If it is determined that it is the first iteration at operation 1112,then the EPRY process proceeds to operation 1113. At operation 1113,using a processor, a differential phase contrast (DPC) deconvolutionprocedure is applied. DPC deconvolution is a partially coherent methodto acquire the quantitative phase of a sample. DPC deconvolution isbased on the assumption that the absorption and phase of a specimenbeing imaged are small such that the specimen's complex transmissionfunction, ψ(x)=exp(−μ(x)+jθ(x)) can be approximated asψ(x)≈1−μ(x)+jθ(x). Under this condition, performing arithmeticoperations on the intensity images captured at different illuminationangles generates multiple-axis DPC images and a transfer functionassociated with the specimen's phase and DPC images. De-convolving thetransfer function from the DPC images results in the quantitative phaseimage of the sample with the spatial frequency information extending to2k₀NA_(obj) in k-space.

The phase of the initial guess is updated with the DPC-deconvolvedquantitative phase as: ψ_(2NA)(x)=|

{Ψ(k)P_(2NA)(x)}|exp(jθ_(DPC)) where Ψ(k) is the high-resolution Fourierspectrum of a sample, P_(2NA) is the low-pass filter with the spatialfrequency extent of 2k₀NA_(obj) in k-space, is Fourier transformoperator, θ_(DPC) is the quantitative phase obtained from DPCdeconvolution, and ψ_(2NA) is the simulated image with its phase updatedwith θ_(DPC.) Unlike intensity image updates in techniques without usinga DPC-updated phase, an update with the phase from DPC deconvolutionrequires use of a pupil function extending to 2NA_(obj) instead of justNA_(obj) because the deconvolved phase contains information up to2NA_(obj) resolution.

Intensity images captured at different angles are used to reconstructthe high resolution Fourier spectrum and the pupil function of themicroscope as done in earlier versions of the FP techniques. DPC phaseneeds to be recalculated at the beginning of each iteration of process1100 because the pupil function of the microscope changes during pupilfunction update procedure.

Further details of an example of an DPC deconvolution process can befound in L. Tian and L. Waller, “Quantitative differential phasecontrast imaging in an LED array microscope,” Opt. Express 23(9),11394-11403 (2015), which is hereby incorporated by reference for thediscussion herein.

In certain embodiments, DPC deconvolution is included in an LFP imagingmethod to address low spatial frequency artifacts or “speckle noise”that may be introduced when a laser light source is used. Such artifactsmay be optionally removed through use of DPC deconvolution because DPCdeconvolution is a partially coherent method and is, therefore, robustto such speckle noise.

By way of illustration, FIG. 12 shows an example of a side-by-sidecomparison of LFP reconstructed images (both phase and amplitude) ofcells that have been enhanced using a DPC procedure and LFPreconstructed images (both phase and amplitude) of the same cells thathave not been enhanced using a DPC procedure. As shown, the images forwhich optional DPC-deconvolution was not used in the reconstructionprocess, the resulting phase image of the sample shows an unevenbackground signal, producing speckles in the cells' phase amplitude. Onthe other hand, in the images for which optional DPC-deconvolution wasused in the reconstruction process, the background is uniform and thecells show similar phase values. Notably, the modification has little tono effect on the amplitude image.

Returning to FIG. 11, in the a^(th) cycle of the inner loop, with theknowledge of the reconstructed S_(a)(u) and P_(a)(u) from the previouscycle of the inner loop, the exit wave at the pupil plane while thesample is illuminated by a wavevector U_(n) can be simulated using:φ_(a)(u)=P_(a)(u)S_(a)(u−U_(n)) with the S_(a)(u) and P_(a)(u) from theprevious cycle. At operation 1116, the processor shifts the samplespectrum according to the illumination angle and multiplies by the pupilfunction according to: φ_(a)(u)=P_(a)(u)S_(a)(u−U_(n)). The pupilfunction comprises both an amplitude and a phase factor. The phasefactor of the pupil function is generally associated with defocus orother aberration associated with the optical system. The amplitude ofthe pupil function is usually associated with the objective lensaperture shape of the optical system. By multiplying the sample spectrumby the pupil function in the Fourier domain, the processor(s) bothfilters the higher-resolution solution by multiplying by the modulus(computed amplitude component) of the pupil function and also multipliesby the phase factor of the pupil function. Multiplying the samplespectrum by the modulus filters the higher-resolution image in theFourier domain for a particular plane wave incidence angle (θ_(x) ^(a),θ_(y) ^(a)) with a wave vector U_(a)=(k_(x), k_(y)). An image capturedwith illumination U_(a) based on the a^(th) illumination incidence angleis referred to in this section as I_(Ua)(r). By multiplying the samplespectrum by the modulus, the processor(s) filters a region from thesample spectrum S(u) in the Fourier domain. In cases with a filteringoptical element in the form of an objective lens, this region takes theform of a circular pupil aperture with a radius of NA*k₀, where k₀equals 2π/λ (the wave number in vacuum), given by the coherent transferfunction of an objective lens. The center of the circular region inFourier space corresponds to the associated illuminating incidence angleof this a^(th) cycle of the inner loop. For an oblique plane waveincidence with a wave vector U_(a)=(k_(x), k_(y)), the region iscentered about a position (k_(x), k_(y)) in the Fourier domain.

At operation 1118, the processor takes the inverse Fourier transform asfollows: φ_(a)(r)=F⁻¹ {φ_(a)(u)}. At operation 1120, the processorimposes an intensity constraint. In this operation 1120, the modulus(computed amplitude component) of the simulated region in Fourier spaceis replaced with the low resolution intensity measurement I_(U) _(a) (r)captured by the radiation detector associated with an illuminationwavevector U_(a). The computed amplitude component is replaced by thesquare-root of the real intensity measurement I_(U) _(a) (r) accordingto:

${\varphi_{a}^{\prime}(r)} = {\sqrt{I_{U_{a}}(r)}{\frac{\varphi_{a}(r)}{{\varphi_{a}(r)}}.}}$

This forms an updated lower resolution image.

At operation 1122, a Fourier transform is applied to the updated lowerresolution image. In this operation, an updated exit wave is calculatedvia a Fourier transform according to: φ′_(a)(u)=

{Φ′_(a)(r)}.

At operation 1124, the processor refreshes the Fourier spectrum guess ofthe higher resolution solution by updating the exit wave data andreplacing data in a corresponding region of the Fourier domain as theupdated exit wave data associated with incidence wave vectorU_(n)=(k_(x), k_(y)). The processor updates the exit wave data using asample spectrum update function. An example of a sample spectrum updatefunction is given by:

$\begin{matrix}{{S_{a + 1}(u)} = {{S_{a}(u)} + {\alpha {\frac{P_{a}^{*}\left( {u + U_{a}} \right)}{{{P_{a}\left( {u + U_{a}} \right)}}_{\max}^{2}}\left\lbrack {{\varphi_{a}^{\prime}\left( {u + U_{a}} \right)} - {\varphi_{a}\left( {u + U_{a}} \right)}} \right\rbrack}}}} & \left( {{Eqn}.\mspace{14mu} 3} \right)\end{matrix}$

By using such a spectrum update function, the updated value of thesample spectrum may be extracted from the difference of the two exitwaves by dividing out the current pupil function. By multiplying withthe conjugates using Eqn. 3 and Eqn. 4, the sample spectrum can beseparated from the pupil function so that the sample spectrum can berefreshed separately from the pupil function. In some cases, acorrection is added to the sample spectrum guess with weightproportional to the intensity of the current pupil function estimate.The constant α adjusts the step size of the update. In one example, α=1.During the cycles of the inner loop, the data is updated as overlappingregions in the Fourier domain.

Concurrently with operation 1124, at operation 1126 the processorrefreshes the guess of the pupil function in the Fourier domain as:P₂₊₁(u). An example of a pupil update function that can be used here isgiven by:

$\begin{matrix}{{P_{a + 1}(u)} = {{{SP}_{a}(u)} + {\beta {\frac{S_{a}^{*}\left( {u + U_{a}} \right)}{{{S_{a}\left( {u + U_{a}} \right)}}_{\max}^{2}}\left\lbrack {{\varphi_{a}^{\prime}(u)} - {\varphi_{a}(u)}} \right\rbrack}}}} & \left( {{Eqn}.\mspace{14mu} 4} \right)\end{matrix}$

The constant β adjusts the step size of the pupil function update andβ=1 is used in this paper. Using this pupil update function, thecorrection of the pupil function is extracted from the difference of thetwo exit waves by dividing out the current sample spectrum estimate, andadded to the current pupil function guess with weight proportional tothe intensity of the current sample spectrum estimate. By multiplying bythe conjugate using Eqn. 4, the pupil function can be separated from thesample spectrum and refreshed separately.

At operation 1128, the processor imposes a pupil function constraint onthe updated pupil function. Imposing the pupil function constraint maysuppress noise. In the example of a microscope system, a physicalcircular aperture stop may be set to define the NA, thus the area in thepupil function that corresponds to the stop should always be zero. Thenon-zero points in the updated pupil function in the regioncorresponding to the stop are caused by the noise in image acquisition,and are set to zero to eliminate the noise.

The inner loop of the method continues to cycle until all N capturedimages in the sequence I_(U) _(a) (r) are used to update the pupil andsample spectrum, at which point an iteration of the outer loop iscomplete. The cycles run from a=0 to N−1. At operation 1130, theprocessor determines whether a=N−1. If the processor determines that adoes not equal N−1, then not all the N captured images have been used.In this case, the loop index a will be incremented at operation 1132,and the method will return to operation 1116 based on the next capturedimage associated with another incidence angle.

If the processor determines that a does equal N−1, the method continuesto operation 1134. If the processor determines that a does not equalN−1, the method continues to operation 1132. At operation 1132, theouter loop index is incremented a=a+1 to the next incidence angle. Themethod will then return to start a new cycle at operation 1116.

At operation 1134, the processor determines whether b=B. If theprocessor determines that b does not equal B, the loop index b will beincremented at operation 1136 to b=b+1 and the loop index a will bereset to 0. The method will then return to start a new iteration atoperation 1112.

If the processor determines that b does equal B, then the iterationsstop and the LFP method continues to operation 1138. At operation 1138,the sample spectrum is inverse Fourier transformed back to the spatialdomain to generate image data for the improved resolution image of thespecimen. Both the image data for the improved resolution image of thespecimen and the pupil function are output of the EPRY process. Thepupil function that we are left with at the end of the operation (i.e.b=B) is the output for the reconstructed pupil function.

III. Experimental Results

An LFP imaging system similar to the LFP imaging system 600 shown inFIG. 6 was used to perform the LFP imaging method 1000 described withrespect to FIGS. 10 and 11 to generate high resolution of a sample asample. FIG. 13A shows a raw image of the sample, and FIG. 13B shows anre-constructed image of the sample generated using the LFP imagingmethod 1000 of FIG. 10, performed using the particular implementation ofLFP imaging system 600 of FIG. 6, described above. The re-constructedimage shown in FIG. 12B includes resolvable features corresponding to1.1 μm periodicity. Since periodicity is calculated by dividing λ (thewavelength of the laser illumination) by NA, a 1.1 μm periodicity isconsistent with the calculated NA of 0.425 of the particularimplementation of LFP imaging system 600 of FIG. 6, which uses 457 nmlaser illumination, as described above. As such, in this particularimplementation, LFP imaging system 600 has been shown to have an NA thatis approximately 4.25 times the NA of collection element 622.

FIG. 14 shows a schematic representation of a phase and amplitudereconstruction process 1400 during the performance of an LFP imagingmethod, according to an implementation. As depicted in FIG. 14, thereconstruction begins at 1410 with a raw image of a sample captured withthe illumination from the center mirror element 816(1) of FIG. 6 as aninitial guess of the sample field. The iteration process starts byforming the sample's quantitative phase image with 2 NA resolution byDPC deconvolution with the initial guess of the pupil function, asdescribed above in the context of FIG. 11. At 1420 of FIG. 14, the phaseof the sample field up to the 2 NA resolution extent is updated, asdescribed above in the context of FIG. 11. In the end of process 1400 ofFIG. 14, the complex sample field 1430 and the pupil function 1440 arereconstructed.

Modifications, additions, or omissions may be made to any of theabove-described embodiments without departing from the scope of thedisclosure. Any of the embodiments described above may include more,fewer, or other features without departing from the scope of thedisclosure. Additionally, the steps of the described features may beperformed in any suitable order without departing from the scope of thedisclosure.

It should be understood that the present invention as described abovecan be implemented in the form of control logic using computer softwarein a modular or integrated manner. Based on the disclosure and teachingsprovided herein, a person of ordinary skill in the art will know andappreciate other ways and/or methods to implement the present inventionusing hardware and a combination of hardware and software.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a CRM, such as a random access memory (RAM), a read onlymemory (ROM), a magnetic medium such as a hard-drive or a floppy disk,or an optical medium such as a CD-ROM. Any such CRM may reside on orwithin a single computational apparatus, and may be present on or withindifferent computational apparatuses within a system or network.

Although the foregoing disclosed embodiments have been described in somedetail to facilitate understanding, the described embodiments are to beconsidered illustrative and not limiting. It will be apparent to one ofordinary skill in the art that certain changes and modifications can bepracticed within the scope of the claims.

One or more features from any embodiment may be combined with one ormore features of any other embodiment without departing from the scopeof the disclosure. Further, modifications, additions, or omissions maybe made to any embodiment without departing from the scope of thedisclosure. The components of any embodiment may be integrated orseparated according to particular needs without departing from the scopeof the disclosure.

As used herein, the conjunction “or” is intended herein in the inclusivesense where appropriate unless otherwise indicated; that is, the phrase“A, B or C” is intended to include the possibilities of A, B, C, A andB, B and C, A and C and A, B and C. Additionally, a phrase referring to“at least one of” a list of items refers to any combination of thoseitems, including single members. As an example, “at least one of: A, B,or C” is intended to cover: A, B, C, A-B, A-C, B-C, and A-B-C.

What is claimed is:
 1. A laser-based Fourier ptychographic imagingsystem, comprising: an angle direction device configured to direct laserlight from a laser light source to a specimen surface at a sequence ofillumination angles; an optical system comprising a collection elementand a focusing element, the collection element configured to receivelight issuing from a specimen when it is located on the specimensurface, wherein the sequence of illumination angles and a numericalaperture of the collection element correspond to overlapping regions ina Fourier domain; and a light detector configured to receive lightfocused by the focusing element from the optical system and acquire aplurality of intensity images of the specimen when it is located on thespecimen surface and illuminated, each intensity image corresponding toa different illumination angle of the sequence of illumination angles.2. The laser-based Fourier ptychographic imaging system of claim 1,further comprising a processor configured to execute instructions foriteratively updating overlapping regions in the Fourier domain with theplurality of intensity images acquired by the light detector.
 3. Thelaser-based Fourier ptychographic imaging system of claim 1, wherein theplurality of intensity images are acquired in less than 1 second.
 4. Thelaser-based Fourier ptychographic imaging system of claim 2, wherein theprocessor is further configured to execute instructions for filteringout low spatial frequency artifacts associated laser light by using adifferential phase contrast deconvolution procedure.
 5. The laser-basedFourier ptychographic imaging system of claim 1, wherein the angledirection device comprises: a plurality of fixed mirrors, each fixedmirror oriented to reflect laser light at one of the plurality ofillumination angles to the specimen surface; and one or more rotatablemirrors configured to reflect laser light from the laser light sourcesequentially to different fixed mirrors of the plurality of fixedmirrors at different sampling times, wherein the plurality of fixedmirrors are oriented to reflect laser light received from the one ormore rotatable mirrors to the specimen surface at the sequence ofillumination angles.
 6. The laser-based Fourier ptychographic imagingsystem of claim 5, wherein the one or more rotatable mirrors comprise: afirst rotatable mirror configured to rotate along a first axis; and asecond rotatable mirror configured to rotate along a second axisorthogonal to the first axis; wherein the first rotatable mirror isconfigured to reflect laser light from the laser light source to thefirst rotatable mirror and the second rotatable mirror is configured toreflect light from the first rotatable mirror to the specimen surface,wherein the first rotatable mirror and second rotatable mirror areconfigured to rotate to direct laser light to different fixed mirrors ofthe plurality of mirrors to direct laser light at the sequence ofillumination angles.
 7. The laser-based Fourier ptychographic imagingsystem of claim 5, wherein the one or more rotatable mirrors comprise adual axis rotatable mirror configured to rotate along two orthogonalaxes to reflect laser light from the laser light source to differentfixed mirrors of the plurality of mirrors to direct laser light at thesequence of illumination angles.
 8. The laser-based Fourierptychographic imaging system of claim 5, wherein the plurality ofmirrors are arranged in concentric rings along a bowl-shaped surface. 9.The laser-based Fourier ptychographic imaging system of claim 5, whereinthe plurality of fixed mirrors are arranged in a flat structure.
 10. Thelaser-based Fourier ptychographic imaging system of claim 5, whereineach fixed mirror of the plurality of mirrors comprises a neutraldensity filter.
 11. The laser-based Fourier ptychographic imaging systemof claim 1, wherein the angle direction device comprises: a plurality ofoptical fibers, each optical fibers having first and second endportions; and one or more optical switches in optical communication withthe laser light source, the one or more optical switches configured toswitch at different sampling times to direct laser light from the laserlight source to the first end portion of different optical fibers of theplurality of optical fibers when the laser light source is activated;wherein the plurality of optical pathways are configured so that eachsecond end portion directs laser light to one of the sequence ofillumination angles when the one or more optical switches is switched tothe corresponding optical fiber and the laser light source is activated.12. The laser-based Fourier ptychographic imaging system of claim 1,wherein in the angle direction device comprises: a movable stage; and anoptical fiber coupled to the movable stage and optically coupled at oneend to the laser light source, wherein the movable stage is configuredor configurable to translate and/or rotate the optical fiber to directlaser light from the other end of the optical fiber to illuminate thespecimen surface at the plurality of illumination angles at thedifferent sampling times.
 13. The laser-based Fourier ptychographicimaging system of claim 12, wherein the movable stage is an X-Y stage,and wherein the optical fiber is coupled to the X-Y stage with aconnector.
 14. The laser-based Fourier ptychographic imaging system ofclaim 1, wherein in the angle direction device comprises: one or morerotatable mirrors; and a lens system, wherein the one or more rotatablemirrors are configurable to direct the laser light from the laser lightsource to the specimen surface through a lens system, the lens systemconfigured such that rotation of the one or more rotatable mirrorscauses the laser light to illuminate the specimen at the sequence ofillumination angles.
 15. The laser-based Fourier ptychographic imagingsystem of claim 18, wherein the lens system comprises a first lens and asecond lens, wherein a back-focal plane of the first lens coincidingwith a focal plane of the second lens, wherein at least one of the firstand second lenses is a f-theta lens.
 16. A laser angle direction devicefor directing laser light at a sequence of illumination angles, thelaser angle direction device comprising: a surface; and a plurality offixed mirrors coupled to the surface, each fixed mirror oriented toreceive laser light and reflect the laser light to one of the pluralityof illumination angles.
 17. The laser angle direction device of claim16, further comprising one or more rotatable mirrors configured toreflect laser light to the plurality of fixed mirrors at differentsampling times.
 18. The laser angle direction device of claim 16,wherein the surface is bowl-shaped.
 19. The angle direction device ofclaim 16, wherein the surface is flat; further comprising a plurality ofstructures, each one of the fixed mirrors attached to one end of thestructures, wherein the plurality of structures are attached to thesurface.
 20. The angle direction device of claim I, wherein each fixedmirror comprises a neutral density filter.
 21. A laser-based Fourierptychographic imaging method employing a laser light source, the methodcomprising: directing laser light from a laser light source to aspecimen surface located at about a sample plane using an angledirection device at a sequence of illumination angles; receiving light,at a light detector, issuing from a sample when the sample is located onthe specimen surface, the light received from an optical system;acquiring a plurality of intensity images based on light received at thelight detector, wherein each intensity image corresponds to one of theillumination angles; and constructing a higher resolution image bysimultaneously updating a pupil function and a sample spectrum, whereinthe sample spectrum is updated in overlapping regions with Fouriertransformed intensity images, wherein each of the overlapping regionscorresponds to one of the plurality of illumination angles.